Pesticidal proteins

ABSTRACT

The subject invention concerns new classes of pesticidally active proteins and the polynucleotide sequences that encode these proteins. In preferred embodiments, these pesticidal proteins have molecular weights of approximately 40-50 kDa and of approximately 10-15 kDa.

CROSS-REFERENCE TO A RELATED APPLICATION

This application is a continuation-in-part of U.S. Ser. No. 09/378,088,filed Aug. 20, 1999 now U.S. Pat. No. 6,372,480, which is acontinuation-in-part of Ser. No. 08/844,188, filed Apr. 18, 1997 nowU.S. Pat. No. 6,127,180, which is a continuation-in-part of Ser. No.08/633,993, filed Apr. 19, 1996, which issued as U.S. Pat. No. 6,083,499on Jul. 4, 2000.

BACKGROUND OF THE INVENTION

Coleopterans are a significant group of agricultural pests which causeextensive damage to crops each year. Examples of coleopteran pestsinclude corn rootworm and alfalfa weevils.

The alfalfa weevil, Hypera postica, and the closely related Egyptianalfalfa weevil, Hypera brunneipennis, are the most important insectpests of alfalfa grown in the United States, with 2.9 million acresinfested in 1984. An annual sum of 20 million dollars is spent tocontrol these pests. The Egyptian alfalfa weevil is the predominantspecies in the southwestern U.S., where it undergoes aestivation (i.e.,hibernation) during the hot summer months. In all other respects, it isidentical to the alfalfa weevil, which predominates throughout the restof the U.S.

The larval stage is the most damaging in the weevil life cycle. Byfeeding at the alfalfa plant's growing tips, the larvae causeskeletonization of leaves, stunting, reduced plant growth, and,ultimately, reductions in yield. Severe infestations can ruin an entirecutting of hay. The adults, also foliar feeders, cause additional, butless significant, damage.

Approximately 10 million acres of U.S. corn are infested with cornrootworn species complex each year. The corn rootworm species complexincludes the northern corn rootworm, Diabrotica barberi, the southerncorn rootworm, D. undecimpunctata howardi, and the western cornrootworm, D. virgifera virgifera. The soil-dwelling larvae of theseDiabrotica species feed on the root of the corn plant, causing lodging.Lodging eventually reduces corn yield and often results in death of theplant. By feeding on cornsilks, the adult beetles reduce pollinationand, therefore, detrimentally affect the yield of corn per plant. Inaddition, adults and larvae of the genus Diabrotica attack cucurbitcrops (cucumbers, melons, squash, etc.) and many vegetable and fieldcrops in commercial production as well as those being grown in homegardens.

Control of corn rootworm has been partially addressed by cultivationmethods, such as crop rotation and the application of high nitrogenlevels to stimulate the growth of an adventitious root system. However,chemical insecticides are relied upon most heavily to guarantee thedesired level of control. Insecticides are either banded onto orincorporated into the soil. Problems associated with the use of somechemical insecticides are environmental contamination and thedevelopment of resistance among the treated insect populations.

The soil microbe Bacillus thuringiensis (B.t.) is a Gram-positive,spore-forming bacterium characterized by parasporal protein inclusions,which can appear microscopically as distinctively shaped crystals.Certain strains of B.t. produce proteins that are toxic to specificorders of pests. Certain B.t. toxin genes have been isolated andsequenced, and recombinant DNA-based B.t. products have been producedand approved for use. In addition, with the use of genetic engineeringtechniques, new approaches for delivering these B.t. endotoxins toagricultural environments are under development, including the use ofplants genetically engineered with endotoxin genes for insect resistanceand the use of stabilized intact microbial cells as B.t. endotoxindelivery vehicles (Gaertner, F. H., L. Kim [1988] TIBTECH6:S4-S7). Thus,isolated B.t. endotoxin genes are becoming commercially valuable.

Commercial use of B.t. pesticides was originally limited to a narrowrange of lepidopteran (caterpillar) pests. Preparations of the sporesand crystals of B. thuringiensis subsp. kurstaki have been used for manyyears as commercial insecticides for lepidopteran pests. For example, B.thuringiensis var. kurstaki HD-1 produces a crystalline δ-endotoxinwhich is toxic to the larvae of a number of lepidopteran insects.

In recent years, however, investigators have discovered B.t. pesticideswith specificities for a much broader range of pests. For example, otherspecies of B.t., namely israelensis and tenebrionis (a.k.a. B.t. M-7,a.k.a. B.t. san diego), have been used commercially to control insectsof the orders Diptera and Coleoptera, respectively (Gaertner, F. H.[1989] “Cellular Delivery Systems for Insecticidal Proteins: Living andNon-Living Microorganisms,” in Controlled Delivery of Crop ProtectionAgents, R. M. Wilkins, ed., Taylor and Francis, New York and London,1990, pp. 245-255). See also Couch, T. L. (1980) “Mosquito Pathogenicityof Bacillus thuringiensis var. israelensis,” Developments in IndustrialMicrobiology 22:61-76; Beegle, C. C., (1978) “Use of EntomogenousBacteria in Agroecosystems,” Developments in Industrial Microbiology20:97-104. Krieg, A., A. M. Huger, G. A. Langenbruch, W. Schnetter(1983) Z. ang. Ent. 96:500-508, describe Bacillus thuringiensis var.tenebrionis, which is reportedly active against two beetles in the orderColeoptera. These are the Colorado potato beetle, Leptinotarsadecemlineata, and Agelastica alni.

Recently, new subspecies of B.t. have been identified, and genesresponsible for active δ-endotoxin proteins have been isolated (Höfte,H., H. R. Whiteley [1989] Microbiological Reviews 52(2):242-255). Höfteand Whiteley classified B.t. crystal protein genes into four majorclasses. The classes were CryI (Lepidoptera-specific), CryII(Lepidoptera- and Diptera-specific), CryIII (Coleoptera-specific), andCryIV (Diptera-specific). The discovery of strains specifically toxic toother pests has been reported. (Feitelson, J. S., J. Payne, L. Kim[1992] Bio/Technology 10:271-275).

The 1989 nomenclature and classification scheme of Höfte and Whiteleyfor crystal proteins was based on both the deduced amino acid sequenceand the host range of the toxin. That system was adapted to coverfourteen different types of toxin genes which were divided into fivemajor classes. As more toxin-genes were discovered, that system startedto become unworkable, as genes with similar sequences were found to havesignificantly different insecticidal specificities. A revisednomenclature scheme has been proposed which is based solely on aminoacid identity (Crickmore et al. [1996] Society for InvertebratePathology, 29th Annual Meeting, 3rd International Colloquium on Bacillusthuringiensis, University of Cordoba, Cordoba, Spain, September 1-6,abstract). The mnemonic “cry” has been retained for all of the toxingenes except cytA and cytB, which remain a separate class. Romannumerals have been exchanged for Arabic numerals in the primary rank,and the parentheses in the tertiary rank have been removed. Currentboundaries represent approximately 95% (tertiary rank), 75% (secondaryrank), and 48% (primary rank) sequence identity. Many of the originalnames have been retained, with the noted exceptions, although a numberhave been reclassified. See also N. Crickmore, D. R. Zeigler, J.Feitelson, E. Schnepf, J. Van Rie, D. Lereclus, J. Baum, and D. H. Dean(1998), “Revisions of the Nomenclature for the Bacillus thuringiensisPesticidal Crystal Proteins,” Microbiology and Molecular Biology ReviewsVol. 62:807-813; and Crickmore, Zeigler, Feitelson, Schnepf, Van Rie,Lereclus, Baum, and Dean, “Bacillus thuringiensis toxin nomenclature”(1999) http://www.biols.susx.ac.uk/Home/Neil_Crickmore/Bt/index.html.That system uses the freely available software applications CLUSTAL Wand PHYLIP. The NEIGHBOR application within the PHYLIP package uses anarithmetic averages (UPGMA) algorithm.

The cloning and expression of a B.t. crystal protein gene in Escherichiacoli has been described in the published literature (Schnepf, H. E., H.R. Whiteley [1981] Proc. Natl. Acad. Sci. USA 78:2893-2897). U.S. Pat.No. 4,448,885 and U.S. Pat. No. 4,467,036 both disclose the expressionof B.t. crystal protein in E. coli.

U.S. Pat. Nos. 4,797,276 and 4,853,331 disclose B. thuringiensis straintenebrionis (a.k.a. M-7, a.k.a. B.t. san diego), which can be used tocontrol coleopteran pests in various environments. U.S. Pat. No.4,918,006 discloses B.t. toxins having activity against Dipterans. U.S.Pat. No. 4,849,217 discloses B.t. isolates which have activity againstthe alfalfa weevil. U.S. Pat. No. 5,208,077 discloses coleopteran-activeBacillus thuringiensis isolates. U.S. Pat. No. 5,632,987 discloses a 130kDa toxin from PS80JJ1 as having activity against corn rootworm. WO94/40162, which is related to the subject application, describes newclasses of proteins that are toxic to corn rootworn. U.S. Pat. No.5,151,363 and U.S. Pat. No. 4,948,734 disclose certain isolates of B.t.which have activity against nematodes.

U.S. Pat. No. 6,083,499 and WO 97/40162 disclose “binary toxins.” Thesubject invention is distinct from mosquitocidal toxins produced byBacillus sphaericus. See EP 454 485; Davidson et al. (1990),“Interaction of the Bacillus sphaericus mosquito larvicidal proteins,”Can. J. Microbio. 36(12):870-8; Baumann et al. (1988), “Sequenceanalysis of the mosquitocidal toxin genes encoding 51.4- and41.9-kilodalton proteins from Bacillus sphaericus 2362 and 2297,” J.Bacteriol. 170:2045-2050; Oei et al. (1992), “Binding of purifiedBacillus sphaericus binary toxin and its deletion derivatives to Culexquinquefasciatus gut: elucidation of functional binding domains,”Journal of General Microbiology 138(7):1515-26.

BRIEF SUMMARY OF THE INVENTION

The subject invention concerns novel materials and methods forcontrolling non-mammalian pests. In a preferred embodiment, the subjectinvention provides materials and methods for the control of coleopteranpests. In more preferred embodiments, the materials and methodsdescribed herein are used to control corn rootworm—most preferablyWestern corn rootworm. Lepidopteran pests (including the European cornborer and Helicoverpa zea) can also be controlled by the pesticidalproteins of the subject invention.

The subject invention advantageously provides polynucleotides andpesticidal proteins encoded by the polynucleotides. In preferredembodiments, a 40-50 kDa protein and a 10-15 kDa protein are usedtogether, with the proteins being pesticidal in combination. Thus, thetwo classes of proteins of the subject invention can be referred to as“binary toxins.” As used herein, the term “toxin” or “pesticidalprotein” includes either class of these proteins. The use of a 40-50 kDaprotein with a 10-15 kDa protein is preferred but not necessarilyrequired. One class of polynucleotide sequences as described hereinencodes proteins which have a full-length molecular weight ofapproximately 40-50 kDa. In a specific embodiment, these proteins have amolecular weight of about 43-47 kDa. A second class of polynucleotidesof the subject invention encodes pesticidal proteins of about 10-15 kDa.In a specific embodiment, these proteins have a molecular weight ofabout 13-14 kDa. It should be clear that each type of toxin/gene is anaspect of the subject invention. In a particularly preferred embodiment,a 40-50 kDa protein of the subject invention is used in combination witha 10-15 kDa protein. Thus, the proteins of the subject invention can beused to augment and/or facilitate the activity of other protein toxins.

The subject invention includes polynucleotides that encode the 40-50 kDaor the 10-15 kDa toxins, polynucleotides that encode portions orfragments of the full length toxins that retain pesticidal activity(preferably when used in combination), and polynucleotides that encodeboth types of toxins. Novel examples of fusion proteins (a 40-50 kDaprotein and a 10-15 kDa protein fused together) and polynucleotides thatencode them are also disclosed herein.

In some embodiments, B.t. toxins useful according to the inventioninclude toxins which can be obtained from the novel B.t. isolatesdisclosed herein. It should be clear that, where 40-50 kDa and 10-15 kDatoxins, for example, are used together, one type of toxin can beobtained from one isolate and the other type of toxin can be obtainedfrom another isolate.

The subject invention also includes the use of variants of theexemplified B.t. isolates and toxins which have substantially the samecoleopteran-active properties as the specifically exemplified B.t.isolates and toxins. Such variant isolates would include, for example,mutants. Procedures for making mutants are well known in themicrobiological art. Ultraviolet light and chemical mutagens such asnitrosoguanidine are used extensively toward this end.

In preferred embodiments, the subject invention concerns plants andplant cells having at least one isolated polynucleotide of the subjectinvention. Preferably, the transgenic plant cells express pesticidaltoxins in tissues consumed by the target pests.

Alternatively, the B.t. isolates of the subject invention, orrecombinant microbes expressing the toxins described herein, can be usedto control pests. In this regard, the invention includes the treatmentof substantially intact B.t. cells, and/or recombinant cells containingthe expressed toxins of the invention, treated to prolong the pesticidalactivity when the substantially intact cells are applied to theenvironment of a target pest. The treated cell acts as a protectivecoating for the pesticidal toxin.

The toxins of the subject invention are oral intoxicants that affect aninsect's midgut cells upon ingestion by the target insect. Thus, byconsuming recombinant host cells, for example, that express the toxins,the target insect thereby contacts the proteins of the subjectinvention, which are toxic to the pest. This results in control of thetarget pest.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B show three exemplary 43-47 kDa pesticidal toxins as wellas a consensus sequence for these pesticidal toxins.

FIG. 2 shows the relationship of the 14 and 45 kDa sequences of PS80JJ1(SEQ ID NOS. 31 and 10).

FIG. 3 shows a comparison of LC₅₀ values from the mixing study ofExample 23.

FIG. 4 shows protein alignments of the 51 and 42 kDa Bacillus sphaericustoxins and genes and the 45 kDa 149B1 toxin and gene.

FIGS. 5A-5C show nucleotide sequence alignments of the 51 and 42 kDaBacillus sphaericus toxins and genes and the 45 kDa 149B1 toxin andgene.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is a 5-amino acid N-terminal sequence of the approximately45 kDa toxin of 80JJ1.

SEQ ID NO:2 is a 25-amino acid N-terminal sequence of the approximately45 kDa toxin of 80JJ1.

SEQ ID NO:3 is a 24-amino acid N-terminal sequence of the approximately14 kDa toxin of 80JJ1.

SEQ ID NO:4 is the N-terminal sequence of the approximately 47 kDa toxinfrom 149B1.

SEQ ID NO:5 is a 50-amino acid N-terminal amino acid sequence for thepurified approximately 14 kDa protein from PS149B1.

SEQ ID NO:6 is the N-terminal sequence of the approximately 47 kDa toxinfrom 167H2.

SEQ ID NO:7 is a 25-amino acid N-terminal sequence for the purifiedapproximately 14 kDa protein from PS167H2.

SEQ ID NO:8 is an oligonucleotide probe for the gene encoding thePS80JJ1 44.3 kDa toxin and is a forward primer for PS149B1 and PS167H2used according to the subject invention.

SEQ ID NO:9 is a reverse primer for PS149B1 and PS167H2 used accordingto the subject invention.

SEQ ID NO:10 is the nucleotide sequence of the gene encoding theapproximately 45 kDa PS80JJ1 toxin.

SEQ ID NO:11 is the amino acid sequence for the approximately 45 kDaPS80JJ1 toxin.

SEQ ID NO:12 is the partial nucleotide sequence of the gene encoding theapproximately 44 kDa PS149B1 toxin.

SEQ ID NO:13 is the partial amino acid sequence for the approximately 44kDa PS149B1 toxin.

SEQ ID NO:14 is the partial nucleotide sequence of the gene encoding theapproximately 44 kDa PS167H2 toxin.

SEQ ID NO:15 is the partial amino acid sequence for the approximately 44kDa PS167H2 toxin.

SEQ ID NO:16 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO:17 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO:18 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO:19 is a peptide sequence used in primer design according tothe subject invention.

SEQ ID NO:20 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO:16.

SEQ ID NO:21 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO:17.

SEQ ID NO:22 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO:18.

SEQ ID NO:23 is a nucleotide sequence corresponding to the peptide ofSEQ ID NO:19.

SEQ ID NO:24 is a reverse primer based on the reverse complement of SEQID NO:22.

SEQ ID NO:25 is a reverse primer based on the reverse complement of SEQID NO:23.

SEQ ID NO:26 is a forward primer based on the PS80JJ1 44.3 kDa toxin.

SEQ ID NO:27 is a reverse primer based on the PS80JJ1 44.3 kDa toxin.

SEQ ID NO:28 is a generic sequence representing a new class of toxinsaccording to the subject invention.

SEQ ID NO:29 is an oligonucleotide probe used according to the subjectinvention.

SEQ ID NO:30 is the nucleotide sequence of the entire genetic locuscontaining open reading frames of both the 14 and 45 kDa PS80JJ1 toxinsand the flanking nucleotide sequences.

SEQ ID NO:31 is the nucleotide sequence of the PS80JJ1 14 kDa toxin openreading frame.

SEQ ID NO:32 is the deduced amino acid sequence of the 14 kDa toxin ofPS80JJ1.

SEQ ID NO:33 is a reverse oligonucleotide primer used according to thesubject invention.

SEQ ID NO:34 is the nucleotide sequence of the entire genetic locuscontaining open reading frames of both the 14 and 44 kDa PS167H2 toxinsand the flanking nucleotide sequences.

SEQ ID NO:35 is the nucleotide sequence of the gene encoding theapproximately 14 kDa PS167H2 toxin.

SEQ ID NO:36 is the amino acid sequence for the approximately 14 kDaPS167H2 toxin.

SEQ ID NO:37 is the nucleotide sequence of the gene encoding theapproximately 44 kDa PS167H2 toxin.

SEQ ID NO:38 is the amino acid sequence for the approximately 44 kDaPS167H2 toxin.

SEQ ID NO:39 is the nucleotide sequence of the entire genetic locuscontaining open reading frames of both the 14 and 44 kDa PS149B1 toxinsand the flanking nucleotide sequences.

SEQ ID NO:40 is the nucleotide sequence of the gene encoding theapproximately 14 kDa PS149B1 toxin.

SEQ ID NO:41 is the amino acid sequence for the approximately 14 kDaPS149B1 toxin.

SEQ ID NO:42 is the nucleotide sequence of the gene encoding theapproximately 44 kDa PS149B1 toxin.

SEQ ID NO:43 is the amino acid sequence for the approximately 44 kDaPS149B1 toxin.

SEQ ID NO:44 is a maize-optimized gene sequence encoding theapproximately 14 kDa toxin of 80JJ1.

SEQ ID NO:45 is a maize-optimized gene sequence encoding theapproximately 44 kDa toxin of 80JJ1.

SEQ ID NO:46 is the DNA sequence of a reverse primer used in Example 15,below.

SEQ ID NO:47 is the DNA sequence of a forward primer (see Example 16).

SEQ ID NO:48 is the DNA sequence of a reverse primer (see Example 16).

SEQ ID NO:49 is the DNA sequence of a forward primer (see Example 16).

SEQ ID NO:50 is the DNA sequence of a reverse primer (see Example 16).

SEQ ID NO:51 is the DNA sequence from PS131W2 which encodes the 14 kDaprotein.

SEQ ID NO:52 is the amino acid sequence of the 14 kDa protein ofPS131W2.

SEQ ID NO:53 is a partial DNA sequence from PS131W2 for the 44 kDaprotein.

SEQ ID NO:54 is a partial amino acid sequence for the 44 kDa protein ofPS131W2.

SEQ ID NO:55 is the DNA sequence from PS158T3 which encodes the 14 kDaprotein.

SEQ ID NO:56 is the amino acid sequence of the 14 kDa protein ofPS158T3.

SEQ ID NO:57 is a partial DNA sequence from PS158T3 for the 44 kDaprotein.

SEQ ID NO:58 is a partial amino acid sequence for the 44 kDa protein ofPS158T3.

SEQ ID NO:59 is the DNA sequence from PS158X10 which encodes the 14 kDaprotein.

SEQ ID NO:60 is the amino acid sequence of the 14 kDa protein ofPS158X10.

SEQ ID NO:61 is the DNA sequence from PS185FF which encodes the 14 kDaprotein.

SEQ ID NO:62 is the amino acid sequence of the 14 kDa protein ofPS185FF.

SEQ ID NO:63 is a partial DNA sequence from PS185FF for the 44 kDaprotein.

SEQ ID NO:64 is a partial amino acid sequence for the 44 kDa protein ofPS185FF.

SEQ ID NO:65 is the DNA sequence from PS185GG which encodes the 14 kDaprotein.

SEQ ID NO:66 is the amino acid sequence of the 14 kDa protein ofPS185GG.

SEQ ID NO:67 is the DNA sequence from PS185GG for the 44 kDa protein.

SEQ ID NO:68 is the amino acid sequence for the 44 kDa protein ofPS185GG.

SEQ ID NO:69 is the DNA sequence from PS185L12 which encodes the 14 kDaprotein.

SEQ ID NO:70 is the amino acid sequence of the 14 kDa protein ofPS185L12.

SEQ ID NO:71 is the DNA sequence from PS185W3 which encodes the 14 kDaprotein.

SEQ ID NO:72 is the amino acid sequence of the 14 kDa protein ofPS185W3.

SEQ ID NO:73 is the DNA sequence from PS186FF which encodes the 14 kDaprotein.

SEQ ID NO:74 is the amino acid sequence of the 14 kDa protein ofPS186FF.

SEQ ID NO:75 is the DNA sequence from PS187F3 which encodes the 14 kDaprotein.

SEQ ID NO:76 is the amino acid sequence of the 14 kDa protein ofPS187F3.

SEQ ID NO:77 is a partial DNA sequence from PS187F3 for the 44 kDaprotein.

SEQ ID NO:78 is a partial amino acid sequence for the 44 kDa protein ofPS187F3.

SEQ ID NO:79 is the DNA sequence from PS187G1 which encodes the 14 kDaprotein.

SEQ ID NO:80 is the amino acid sequence of the 14 kDa protein ofPS187G1.

SEQ ID NO:81 is a partial DNA sequence from PS187G1 for the 44 kDaprotein.

SEQ ID NO:82 is a partial amino acid sequence for the 44 kDa protein ofPS187G1.

SEQ ID NO:83 is the DNA sequence from PS187L14 which encodes the 14 kDaprotein.

SEQ ID NO:84 is the amino acid sequence of the 14 kDa protein ofPS187L14.

SEQ ID NO:85 is a partial DNA sequence from PS187L14 for the 44 kDaprotein.

SEQ ID NO:86 is a partial amino acid sequence for the 44 kDa protein ofPS187L14.

SEQ ID NO:87 is the DNA sequence from PS187Y2 which encodes the 14 kDaprotein.

SEQ ID NO:88 is the amino acid sequence of the 14 kDa protein ofPS187Y2.

SEQ ID NO:89 is a partial DNA sequence from PS187Y2 for the 44 kDaprotein.

SEQ ID NO:90 is a partial amino acid sequence for the 44 kDa protein ofPS187Y2.

SEQ ID NO:91 is the DNA sequence from PS201G which encodes the 14 kDaprotein.

SEQ ID NO:92 is the amino acid sequence of the 14 kDa protein of PS201G.

SEQ ID NO:93 is the DNA sequence from PS201HH which encodes the 14 kDaprotein.

SEQ ID NO:94 is the amino acid sequence of the 14 kDa protein ofPS201HH.

SEQ ID NO:95 is the DNA sequence from PS201L3 which encodes the 14 kDaprotein.

SEQ ID NO:96 is the amino acid sequence of the 14 kDa protein ofPS201L3.

SEQ ID NO:97 is the DNA sequence from PS204C3 which encodes the 14 kDaprotein.

SEQ ID NO:98 is the amino acid sequence of the 14 kDa protein ofPS204C3.

SEQ ID NO:99 is the DNA sequence from PS204G4 which encodes the 14 kDaprotein.

SEQ ID NO:100 is the amino acid sequence of the 14 kDa protein ofPS204G4.

SEQ ID NO:101 is the DNA sequence from PS204I11 which encodes the 14 kDaprotein.

SEQ ID NO:102 is the amino acid sequence of the 14 kDa protein ofPS204I11.

SEQ ID NO:103 is the DNA sequence from PS204J7 which encodes the 14 kDaprotein.

SEQ ID NO:104 is the amino acid sequence of the 14 kDa protein ofPS204J7.

SEQ ID NO:105 is the DNA sequence from PS236B6 which encodes the 14 kDaprotein.

SEQ ID NO:106 is the amino acid sequence of the 14 kDa protein ofPS236B6.

SEQ ID NO:107 is the DNA sequence from PS242K10 which encodes the 14 kDaprotein.

SEQ ID NO:108 is the amino acid sequence of the 14 kDa protein ofPS242K10.

SEQ ID NO:109 is a partial DNA sequence from PS242K10 for the 44 kDaprotein.

SEQ ID NO:110 is a partial amino acid sequence for the 44 kDa protein ofPS242K10.

SEQ ID NO:111 is the DNA sequence from PS246P42 which encodes the 14 kDaprotein.

SEQ ID NO:112 is the amino acid sequence of the 14 kDa protein ofPS246P42.

SEQ ID NO:113 is the DNA sequence from PS69Q which encodes the 14 kDaprotein.

SEQ ID NO:114 is the amino acid sequence of the 14 kDa protein of PS69Q.

SEQ ID NO:115 is the DNA sequence from PS69Q for the 44 kDa protein.

SEQ ID NO:116 is the amino acid sequence for the 44 kDa protein ofPS69Q.

SEQ ID NO:117 is the DNA sequence from KB54 which encodes the 14 kDaprotein.

SEQ ID NO:118 is the amino acid sequence of the 14 kDa protein of KB54.

SEQ ID NO:119 is the DNA sequence from KR1209 which encodes the 14 kDaprotein.

SEQ ID NO:120 is the amino acid sequence of the 14 kDa protein ofKR1209.

SEQ ID NO:121 is the DNA sequence from KR1369 which encodes the 14 kDaprotein.

SEQ ID NO:122 is the amino acid sequence of the 14 kDa protein ofKR1369.

SEQ ID NO:123 is the DNA sequence from KR589 which encodes the 14 kDaprotein.

SEQ ID NO:124 is the amino acid sequence of the 14 kDa protein of KR589.

SEQ ID NO:125 is a partial DNA sequence from KR589 for the 44 kDaprotein.

SEQ ID NO:126 is a partial amino acid sequence for the 44 kDa protein ofKR589.

SEQ ID NO:127 is a polynucleotide sequence for a gene designated149B1-15-PO, which is optimized for expression in Zea mays. This geneencodes an approximately 15 kDa toxin obtainable from PS149B1 that isdisclosed in WO 97/40162.

SEQ ID NO:128 is a polynucleotide sequence for a gene designated149B1-45-PO, which is optimized for expression in Zea mays. This geneencodes an approximately 45 kDa toxin obtainable from PS149B1 that isdisclosed in WO 97/40162.

SEQ ID NO:129 is a polynucleotide sequence for a gene designated80JJ1-15-PO7, which is optimized for expression in maize. This is analternative gene that encodes an approximately 15 kDa toxin.

SEQ ID NO:130 is an amino acid sequence for a toxin encoded by the genedesignated 80JJ1-15-PO7.

SEQ ID NO:131 is an oligonucleotide primer (15kfor1) used according tothe subject invention (see Example 20).

SEQ ID NO:132 is an oligonucleotide primer (45krev6) used according tothe subject invention (see Example 20).

SEQ ID NO:133 is the DNA sequence from PS201L3 which encodes the 14 kDaprotein.

SEQ ID NO:134 is the amino acid sequence of the 14 kDa protein ofPS201L3.

SEQ ID NO:135 is a partial DNA sequence from PS201L3 for the 44 kDaprotein.

SEQ ID NO:136 is a partial amino acid sequence for the 44 kDa protein ofPS201L3.

SEQ ID NO:137 is the DNA sequence from PS187G1 which encodes the 14 kDaprotein.

SEQ ID NO:138 is the amino acid sequence of the 14 kDa protein ofPS187G1.

SEQ ID NO:139 is the DNA sequence from PS187G1 which encodes the 44 kDaprotein.

SEQ ID NO:140 is the amino acid sequence of the 44 kDa protein ofPS187G1.

SEQ ID NO:141 is the DNA sequence from PS201HH2 which encodes the 14 kDaprotein.

SEQ ID NO:142 is the amino acid sequence of the 14 kDa protein ofPS201HH2.

SEQ ID NO:143 is a partial DNA sequence from PS201HH2 for the 44 kDaprotein.

SEQ ID NO:144 is a partial amino acid sequence for the 44 kDa protein ofPS201HH2.

SEQ ID NO:145 is the DNA sequence from KR1369 which encodes the 14 kDaprotein.

SEQ ID NO:146 is the amino acid sequence of the 14 kDa protein ofKR1369.

SEQ ID NO:147 is the DNA sequence from KR1369 which encodes the 44 kDaprotein.

SEQ ID NO:148 is the amino acid sequence of the 44 kDa protein ofKR1369.

SEQ ID NO:149 is the DNA sequence from PS137A which encodes the 14 kDaprotein.

SEQ ID NO:150 is the amino acid sequence of the 14 kDa protein ofPS137A.

SEQ ID NO:151 is the DNA sequence from PS201V2 which encodes the 14 kDaprotein.

SEQ ID NO:152 is the amino acid sequence of the 14 kDa protein ofPS201V2.

SEQ ID NO:153 is the DNA sequence from PS207C3 which encodes the 14 kDaprotein.

SEQ ID NO:154 is the amino acid sequence of the 14 kDa protein ofPS207C3.

SEQ ID NO:155 is an oligonucleotide primer (F1new) for use according tothe subject invention (see Example 22).

SEQ ID NO:156 is an oligonucleotide primer (R1new) for use according tothe subject invention (see Example 22).

SEQ ID NO:157 is an oligonucleotide primer (F2new) for use according tothe subject invention (see Example 22).

SEQ ID NO:158 is an oligonucleotide primer (R2new) for use according tothe subject invention (see Example 22).

SEQ ID NO:159 is an approximately 58 kDa fusion protein.

SEQ ID NO:160 is a fusion gene encoding the protein of SEQ ID NO:159.

SEQ ID NO:161 is primer 45 kD5′ for use according to the subjectinvention (see Example 27).

SEQ ID NO:162 is primer 45 kD3′rc for use according to the subjectinvention (see Example 27).

SEQ ID NO:163 is primer 45 kD5′01 for use according to the subjectinvention (see Example 27).

SEQ ID NO:164 is primer 45 kD5′02 for use according to the subjectinvention (see Example 27).

SEQ ID NO:165 is primer 45 kD3′03 for use according to the subjectinvention (see Example 27).

SEQ ID NO:166 is primer 45 kD3′04 for use according to the subjectinvention (see Example 27).

DETAILED DISCLOSURE OF THE INVENTION

The subject invention concerns two new classes of polynucleotidesequences as well as the novel pesticidal proteins encoded by thesepolynucleotides. In one embodiment, the proteins have a full-lengthmolecular weight of approximately 40-50 kDa. In specific embodimentsexemplified herein, these proteins have a molecular weight of about43-47 kDa. In a second embodiment, the pesticidal proteins have amolecular weight of approximately 10-15 kDa. In specific embodimentsexemplified herein, these proteins have a molecular weight of about13-14 kDa.

In preferred embodiments, a 40-50 kDa protein and a 10-15 kDa proteinare used together, and the proteins are pesticidal in combination. Thus,the two classes of proteins of the subject invention can be referred toas “binary toxins.” As used herein, the term “toxin” includes eitherclass of pesticidal proteins. The subject invention concernspolynucleotides which encode either the 40-50 kDa or the 10-15 kDatoxins, polynucleotides which encode portions or fragments of the fulllength toxins that retain pesticidal activity when used in combination,and polynucleotide sequences which encode both types of toxins. In apreferred embodiment, these toxins are active against coleopteran pests,more preferably corn rootworn, and most preferably Western cornrootworm. Lepidopteran pests can also be targeted.

Certain specific toxins are exemplified herein. For toxins having aknown amino acid sequence, the molecular weight is also known. Thoseskilled in the art will recognize that the apparent molecular weight ofa protein as determined by gel electrophoresis will sometimes differfrom the true molecular weight. Therefore, reference herein to, forexample, a 45 kDa protein or a 14 kDa protein is understood to refer toproteins of approximately that size even if the true molecular weight issomewhat different.

The subject invention concerns not only the polynucleotides that encodethese classes of toxins, but also the use of these polynucleotides toproduce recombinant hosts which express the toxins. In a further aspect,the subject invention concerns the combined use of an approximately40-50 kDa toxin of the subject invention together with an approximately10-15 kDa toxin of the subject invention to achieve highly effectivecontrol of pests, including coleopterans such as corn rootworm. Forexample, the roots of one plant can express both types of toxins.

Thus, control of pests using the isolates, toxins, and genes of thesubject invention can be accomplished by a variety of methods known tothose skilled in the art. These methods include, for example, theapplication of B.t. isolates to the pests (or their location), theapplication of recombinant microbes to the pests (or their locations),and the transformation of plants with genes which encode the pesticidaltoxins of the subject invention. Microbes for use according to thesubject invention may be, for example, B.t., E. coli, and/orPseudomonas. Recombinant hosts can be made by those skilled in the artusing standard techniques. Materials necessary for these transformationsare disclosed herein or are otherwise readily available to the skilledartisan. Control of insects and other pests such as nematodes and mitescan also be accomplished by those skilled in the art using standardtechniques combined with the teachings provided herein.

The new classes of toxins and polynucleotide sequences provided here aredefined according to several parameters. One critical characteristic ofthe toxins described herein is pesticidal activity. In a specificembodiment, these toxins have activity against coleopteran pests.Anti-lepidopteran-active toxins are also embodied. The toxins and genesof the subject invention can be further defined by their amino acid andnucleotide sequences. The sequences of the molecules within each novelclass can be identified and defined in terms of their similarity oridentity to certain exemplified sequences as well as in terms of theability to hybridize with, or be amplified by, certain exemplifiedprobes and primers. The classes of toxins provided herein can also beidentified based on their immunoreactivity with certain antibodies andbased upon their adherence to a generic formula.

It should be apparent to a person skilled in this art that genesencoding pesticidal proteins according to the subject invention can beobtained through several means. The specific genes exemplified hereinmay be obtained from the isolates deposited at a culture depository asdescribed herein. These genes, and toxins, of the subject invention canalso be constructed synthetically, for example, by the use of a genesynthesizer.

The sequence of three exemplary 45 kDa toxins are provided as SEQ IDNOS:11, 43, and 38. In preferred embodiments, toxins of this class havea sequence which conforms to the generic sequence presented as SEQ IDNO:28. In preferred embodiments, the toxins of this class will conformto the consensus sequence shown in FIG. 1.

With the teachings provided herein, one skilled in the art could readilyproduce and use the various toxins and polynucleotide sequences of thenovel classes described herein.

Microorganisms useful according to the subject invention have beendeposited in the permanent collection of the Agricultural ResearchService Patent Culture Collection (NRRL), Northern Regional ResearchCenter, 1815 North University Street, Peoria, Ill. 61604, USA. Theculture repository numbers of the deposited strains are as follows:

Culture Repository No. Deposit Date B.t. strain PS80JJ1 NRRL B-18679July 17, 1990 B.t. strain PS149B1 NRRL B-21553 March 28, 1996 B.t.strain PS167H2 NRRL B-21554 March 28, 1996 E. coli NM522(pMYC2365) NRRLB-21170 Jan. 5, 1994 E. coli NM522(pMYC2382) NRRL B-21329 Sept. 28, 1994E. coli NM522(pMYC2379) NRRL B-21155 Nov. 3, 1993 E. coliNM522(pMYC2421) NRRL B-21555 March 28, 1996 E. coli NM522(pMYC2427) NRRLB-21672 March 26, 1997 E. coli NM522(pMYC2429) NRRL B-21673 March 26,1997 E. coli NM522(pMYC2426) NRRL B-21671 March 26, 1997 B.t. strainPS185GG NRRL B-30175 Aug. 19, 1999 B.t. strain PS187G1 NRRL B-30185 Aug.19, 1999 B.t. strain PS187Y2 NRRL B-30187 Aug. 19, 1999 B.t. strainPS201G NRRL B-30188 Aug. 19, 1999 B.t. strain PS201HH2 NRRL B-30190 Aug.19, 1999 B.t. strain PS242K10 NRRL B-30195 Aug. 19, 1999 B.t. strainPS69Q NRRL B-30175 Aug. 19, 1999 B.t. strain KB54A1-6 NRRL B-30197 Aug.19, 1999 B.t. strain KR589 NRRL B-30198 Aug. 19, 1999 B.t. strainPS185L12 NRRL B-30179 Aug. 19, 1999 B.t. strain PS185W3 NRRL B-30180Aug. 19, 1999 B.t. strain PS187L14 NRRL B-30186 Aug. 19, 1999 B.t.strain PS186FF NRRL B-30182 Aug. 19, 1999 B.t. strain PS131W2 NRRLB-30176 Aug. 19, 1999 B.t. strain PS158T3 NRRL B-30177 Aug. 19, 1999B.t. strain PS158X10 NRRL B-30178 Aug. 19, 1999 B.t. strain PS185FF NRRLB-30182 Aug. 19, 1999 B.t. strain PS187F3 NRRL B-30184 Aug. 19, 1999B.t. strain PS201L3 NRRL B-30189 Aug. 19, 1999 B.t. strain PS204C3 NRRLB-30191 Aug. 19, 1999 B.t. strain PS204G4 NRRL B-18685 July 17, 1990B.t. strain PS204I11 NRRL B-30192 Aug. 19, 1999 B.t. strain PS204J7 NRRLB-30193 Aug. 19, 1999 B.t. strain PS236B6 NRRL B-30194 Aug. 19, 1999B.t. strain PS246P42 NRRL B-30196 Aug. 19, 1999 B.t. strain KR1209 NRRLB-30199 Aug. 19, 1999 B.t. strain KR1369 NRRL B-30200 Aug. 19, 1999 B.t.strain MR1506 NRRL B-30298 June 1, 2000 B.t. strain MR1509 NRRL B-30330Aug. 8, 2000 B.t. strain MR1510 NRRL B-30331 Aug. 8, 2000 P.f. strainMR1607 NRRL B-30332 Aug. 8, 2000

The PS80JJ1 isolate is available to the public by virtue of the issuanceof U.S. Pat. No. 5,151,363 and other patents.

A further aspect of the subject invention concerns novel isolates andthe toxins and genes obtainable from these isolates. Novel isolates havebeen deposited and are included in the above list. These isolates havebeen deposited under conditions that assure that access to the cultureswill be available during the pendency of this patent application to onedetermined by the Commissioner of Patents and Trademarks to be entitledthereto under 37 CFR 1.14 and 35 U.S.C. 122. The deposits are availableas required by foreign patent laws in countries wherein counterparts ofthe subject application, or its progeny, are filed. However, it shouldbe understood that the availability of a deposit does not constitute alicense to practice the subject invention in derogation of patent rightsgranted by governmental action.

Further, the subject culture deposits will be stored and made availableto the public in accord with the provisions of the Budapest Treaty forthe Deposit of Microorganisms, i.e., they will be stored with all thecare necessary to keep them viable and uncontaminated for a period of atleast five years after the most recent request for the furnishing of asample of a deposit, and in any case, for a period of at least 30(thirty) years after the date of deposit or for the enforceable life ofany patent which may issue disclosing the cultures. The depositoracknowledges the duty to replace the deposit(s) should the depository beunable to furnish a sample when requested, due to the condition of thedeposit(s). All restrictions on the availability to the public of thesubject culture deposits will be irrevocably removed upon the grantingof a patent disclosing them.

Following is a table which provides characteristics of certain B.t.isolates that are useful according to the subject invention.

TABLE 1 Description of B.t. strains toxic to coleopterans CrystalDescrip- Approx. Sero- NRRL Deposit Culture tion MW (kDa) type DepositDate PS80JJ1 multiple 130, 90, 47, 4a4b, B- July 17, 1990 attached 37,14 sotto 18679 PS149B1 130, 47, 14 B- March 28, 1996 21553 PS167H2 70,47, 14 B- March 28, 1996 23554

Other isolates of the subject invention can also be characterized interms of the shape and location of toxin inclusions.

Toxins, genes, and probes. The polynucleotides of the subject inventioncan be used to form complete “genes” to encode proteins or peptides in adesired host cell. For example, as the skilled artisan would readilyrecognize, some of the polynucleotides in the attached sequence listingare shown without stop codons. Also, the subject polynucleotides can beappropriately placed under the control of a promoter in a host ofinterest, as is readily known in the art.

As the skilled artisan would readily recognize, DNA typically exists ina double-stranded form. In this arrangement, one strand is complementaryto the other strand and vice versa. As DNA is replicated in a plant (forexample) additional, complementary strands of DNA are produced. The“coding strand” is often used in the art to refer to the strand thatbinds with the anti-sense strand. The mRNA is transcribed from the“anti-sense” strand of DNA. The “sense” or “coding” strand has a seriesof codons (a codon is three nucleotides that can be read three-at-a-timeto yield a particular amino acid) that can be read as an open readingframe (ORF) to form a protein or peptide of interest. In order toexpress a protein in vivo, a strand of DNA is typically transcribed intoa complementary strand of mRNA which is used as the template for theprotein. Thus, the subject invention includes the use of the exemplifiedpolynucleotides shown in the attached sequence listing and/or thecomplementary strands. RNA and PNA (peptide nucleic acids) that arefunctionally equivalent to the exemplified DNA are included in thesubject invention.

Toxins and genes of the subject invention can be identified and obtainedby using oligonucleotide probes, for example. These probes aredetectable nucleotide sequences which may be detectable by virtue of anappropriate label or may be made inherently fluorescent as described inInternational Application No. WO 93/16094. The probes (and thepolynucleotides of the subject invention) may be DNA, RNA, or PNA. Inaddition to adenine (A), cytosine (C), guanine (G), thymine (T), anduracil (U; for RNA molecules), synthetic probes (and polynucleotides) ofthe subject invention can also have inosine (a neutral base capable ofpairing with all four bases; sometimes used in place of a mixture of allfour bases in synthetic probes). Thus, where a synthetic, degenerateoligonucleotide is referred to herein, and “n” is used generically, “n”can be G, A, T, C, or inosine. Ambiguity codes as used herein are inaccordance with standard IUPAC naming conventions as of the filing ofthe subject application (for example, R means A or G, Y means C or T,etc.) As is well known in the art, if the probe molecule and nucleicacid sample hybridize by forming a strong bond between the twomolecules, it can be reasonably assumed that the probe and sample havesubstantial homology/similarity/identity. Preferably, hybridization isconducted under stringent conditions by techniques well-known in theart, as described in, for example, Keller, G. H., M. M. Manak (1987) DNAProbes, Stockton Press, New York, N.Y., pp. 169-170. For example, asstated therein, high stringency conditions can be achieved by firstwashing with 2×SSC (Standard Saline Citrate)/0.1% SDS (Sodium DodecylSulfate) for 15 minutes at room temperature. Two washes are typicallyperformed. Higher stringency can then be achieved by lowering the saltconcentration and/or by raising the temperature. For example, the washdescribed above can be followed by two washings with 0.1×SSC/0.1% SDSfor 15 minutes each at room temperature followed by subsequent washeswith 0.1×SSC/0.1% SDS for 30 minutes each at 55° C. These temperaturescan be used with other hybridization and wash protocols set forth hereinand as would be known to one skilled in the art (SSPE can be used as thesalt instead of SSC, for example). The 2×SSC/0.1% SDS can be prepared byadding 50 ml of 20×SSC and 5 ml of 10% SDS to 445 ml of water. 20×SSCcan be prepared by combining NaCl (175.3 g/0.150 M), sodium citrate(88.2 g/0.015 M), and water to 1 liter, followed by adjusting pH to 7.0with 10 N NaOH. 10% SDS can be prepared by dissolving 10 g of SDS in 50ml of autoclaved water, diluting to 100 ml, and aliquotting.

Detection of the probe provides a means for determining in a knownmanner whether hybridization has occurred. Such a probe analysisprovides a rapid method for identifying toxin-encoding genes of thesubject invention. The nucleotide segments which are used as probesaccording to the invention can be synthesized using a DNA synthesizerand standard procedures. These nucleotide sequences can also be used asPCR primers to amplify genes of the subject invention.

Hybridization characteristics of a molecule can be used to definepolynucleotides of the subject invention. Thus the subject inventionincludes polynucleotides (and/or their complements, preferably theirfull complements) that hybridize with a polynucleotide exemplifiedherein (such as the DNA sequences included in SEQ ID NOs:46-166).

As used herein “stringent” conditions for hybridization refers toconditions which achieve the same, or about the same, degree ofspecificity of hybridization as the conditions employed by the currentapplicants. Specifically, hybridization of immobilized DNA on Southernblots with ³²P-labeled gene-specific probes was performed by standardmethods (Maniatis, T., E. F. Fritsch, J. Sambrook [1982] MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y.). In general, hybridization and subsequent washes werecarried out under stringent conditions that allowed for detection oftarget sequences (with homology to the PS80JJ1 toxin genes, forexample). For double-stranded DNA gene probes, hybridization was carriedout overnight at 20-25° C. below the melting temperature (Tm) of the DNAhybrid in 6×SSPE, 5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denaturedDNA. The melting temperature is described by the following formula(Beltz, G. A., K. A. Jacobs, T. H. Eickbush, P. T. Cherbas, and F. C.Kafatos [1983] Methods of Enzymology, R. Wu, L. Grossman and K. Moldave[eds.] Academic Press, New York 100:266-285):

 Tm=81.5° C.+16.6 Log[Na+]+0.41(%G+C)−0.61(%formamide)−600/length ofduplex in base pairs.

Washes are typically carried out as follows:

(1) Twice at room temperature for 15 minutes in 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at Tm−20° C. for 15 minutes in 0.2×SSPE, 0.1% SDS (moderatestringency wash).

For oligonucleotide probes, hybridization was carried out overnight at10-20° C. below the melting temperature (Tm) of the hybrid in 6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA. Tm foroligonucleotide probes was determined by the following formula:

Tm(° C.)=2(number T/A base pairs)+4(number G/C base pairs)

(Suggs, S. V., T. Miyake, E. H. Kawashime, M. J. Johnson, K. Itakura,and R. B. Wallace [1981] ICN-UCLA Symp. Dev. Biol. Using Purified Genes,D. D. Brown [ed.], Academic Press, New York, 23:683-693).

Washes were typically carried out as follows:

(1) Twice at room temperature for 15 minutes 1×SSPE, 0.1% SDS (lowstringency wash).

(2) Once at the hybridization temperature for 15 minutes in 1×SSPE, 0.1%SDS (moderate stringency wash).

Toxins obtainable from isolates PS149B1, PS167H2, and PS80JJ1 have beencharacterized as having have at least one of the followingcharacteristics (novel toxins of the subject invention can be similarlycharacterized with this and other identifying information set forthherein):

(a) said toxin is encoded by a nucleotide sequence which hybridizesunder stringent conditions with a nucleotide sequence selected from thegroup consisting of: DNA which encodes SEQ ID NO:2, DNA which encodesSEQ ID NO:4, DNA which encodes SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,DNA which encodes SEQ ID NO:11, SEQ ID NO:12, DNA which encodes SEQ IDNO:13, SEQ ID NO:14, DNA which encodes SEQ ID NO:15, DNA which encodesSEQ ID NO:16, DNA which encodes SEQ ID NO:17, DNA which encodes SEQ IDNO:18, DNA which encodes SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ IDNO:27, DNA which encodes a pesticidal portion of SEQ ID NO:28, SEQ IDNO:37, DNA which encodes SEQ ID NO:38, SEQ ID NO:42, and DNA whichencodes SEQ ID NO:43;

(b) said toxin immunoreacts with an antibody to an approximately 40-50kDa pesticidal toxin, or a fragment thereof, from a Bacillusthuringiensis isolate selected from the group consisting of PS80JJ1having the identifying characteristics of NRRL B-18679, PS149B1 havingthe identifying characteristics of NRRL B-21553, and PS167H2 having theidentifying characteristics of NRRL B-21554;

(c) said toxin is encoded by a nucleotide sequence wherein a portion ofsaid nucleotide sequence can be amplified by PCR using a primer pairselected from the group consisting of SEQ ID NOs:20 and 24 to produce afragment of about 495 bp, SEQ ID NOs:20 and 25 to produce a fragment ofabout 594 bp, SEQ ID NOs:21 and 24 to produce a fragment of about 471bp, and SEQ ID NOs:21 and 25 to produce a fragment of about 580 bp;

(d) said toxin comprises a pesticidal portion of the amino acid sequenceshown in SEQ ID NO:28;

(e) said toxin comprises an amino acid sequence which has at least about60% homology with a pesticidal portion of an amino acid sequenceselected from the group consisting of SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:38, and SEQ ID NO:43;

(f) said toxin is encoded by a nucleotide sequence which hybridizesunder stringent conditions with a nucleotide sequence selected from thegroup consisting of DNA which encodes SEQ ID NO:3, DNA which encodes SEQID NO:5, DNA which encodes SEQ ID NO:7, DNA which encodes SEQ ID NO:32,DNA which encodes SEQ ID NO:36, and DNA which encodes SEQ ID NO:41;

(g) said toxin immunoreacts with an antibody to an approximately 10-15kDa pesticidal toxin, or a fragment thereof, from a Bacillusthuringiensis isolate selected from the group consisting of PS80JJ1having the identifying characteristics of NRRL B-18679, PS149B1 havingthe identifying characteristics of NRRL B-21553, and PS167H2 having theidentifying characteristics of NRRL B-21554;

(h) said toxin is encoded by a nucleotide sequence wherein a portion ofsaid nucleotide sequence can be amplified by PCR using the primer pairof SEQ ID NO:29 and SEQ ID NO:33; and

(i) said toxin comprises an amino acid sequence which has at least about60% homology with an amino acid sequence selected from the groupconsisting of SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, pesticidal portionsof SEQ ID NO:32, pesticidal portions of SEQ ID NO:36, and pesticidalportions of SEQ ID NO:41.

Modification of genes and toxins. The genes and toxins useful accordingto the subject invention include not only the specifically exemplifiedfull-length sequences, but also portions and/or fragments (includinginternal and/or terminal deletions compared to the full-lengthmolecules) of these sequences, variants, mutants, chimerics, and fusionsthereof. Proteins of the subject invention can have substituted aminoacids so long as they retain the characteristic pesticidal activity ofthe proteins specifically exemplified herein. “Variant” genes havenucleotide sequences which encode the same toxins or which encode toxinshaving pesticidal activity equivalent to an exemplified protein. As usedherein, the term “equivalent toxins” refers to toxins having the same oressentially the same biological activity against the target pests as theexemplified toxins. As used herein, reference to “essentially the same”sequence refers to sequences which have amino acid substitutions,deletions, additions, or insertions which do not materially affectpesticidal activity. Fragments retaining pesticidal activity are alsoincluded in this definition. Fragments and equivalents which retain thepesticidal activity of the exemplified toxins would be within the scopeof the subject invention.

Equivalent toxins and/or genes encoding these equivalent toxins can bederived from wild-type or recombinant B.t. isolates and/or from otherwild-type or recombinant organisms using the teachings provided herein.Other Bacillus species, for example, can be used as source isolates.

Variations of genes may be readily constructed using standard techniquesfor making point mutations, for example. Also, U.S. Pat. No. 5,605,793,for example, describes methods for generating additional moleculardiversity by using DNA reassembly after random fragmentation. Variantgenes can be used to produce variant proteins; recombinant hosts can beused to produce the variant proteins. Fragments of full-length genes canbe made using commercially available exonucleases or endonucleasesaccording to standard procedures. For example, enzymes such as Bal31 orsite-directed mutagenesis can be used to systematically cut offnucleotides from the ends of these genes. Also, genes which encodeactive fragments may be obtained using a variety of restriction enzymes.Proteases may be used to directly obtain active fragments of thesetoxins.

There are a number of methods for obtaining the pesticidal toxins of theinstant invention. For example, antibodies to the pesticidal toxinsdisclosed and claimed herein can be used to identify and isolate othertoxins from a mixture of proteins. Specifically, antibodies may beraised to the portions of the toxins which are most constant and mostdistinct from other B.t. toxins. These antibodies can then be used tospecifically identify equivalent toxins with the characteristic activityby immunoprecipitation, enzyme linked immunosorbent assay (ELISA), orwestern blotting. Antibodies to the toxins disclosed herein, or toequivalent toxins, or to fragments of these toxins, can readily beprepared using standard procedures. The genes which encode these toxinscan then be obtained from the source microorganism.

Because of the redundancy of the genetic code, a variety of differentDNA sequences can encode the amino acid sequences disclosed herein. Itis well within the skill of a person trained in the art to create thesealternative DNA sequences encoding the same, or essentially the same,toxins. These variant DNA sequences are within the scope of the subjectinvention.

Certain toxins of the subject invention have been specificallyexemplified herein. Since these toxins are merely exemplary of thetoxins of the subject invention, it should be readily apparent that thesubject invention comprises variant or equivalent toxins (and nucleotidesequences coding for equivalent toxins) having the same or similarpesticidal activity of the exemplified toxin. Equivalent toxins willhave amino acid similarity (and/or homology) with an exemplified toxin.The amino acid identity will typically be greater than 60%, preferablygreater than 75%, more preferably greater than 80%, even more preferablygreater than 90%, and can be greater than 95%. Preferred polynucleotidesand proteins of the subject invention can also be defined in terms ofmore particular identity and/or similarity ranges. For example, theidentity and/or similarity can be 49, 50, 51, 52, 53, 54, 55, 56, 57,58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, or 99% as compared to a sequence exemplified herein.Unless otherwise specified, as used herein percent sequence identityand/or similarity of two nucleic acids is determined using the algorithmof Karlin and Altschul (1990), Proc. Natl. Acad. Sci. USA 87:2264-2268,modified as in Karlin and Altschul (1993), Proc. Natl. Acad. Sci. USA90:5873-5877. Such an algorithm is incorporated into the NBLAST andXBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215:402-410.BLAST nucleotide searches are performed with the NBLAST program,score=100, wordlength=12, to obtain nucleotide sequences with thedesired percent sequence identity. To obtain gapped alignments forcomparison purposes, Gapped BLAST is used as described in Altschul etal. (1997), Nucl. Acids Res. 25:3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(NBLAST and XBLAST) are used. See http://www.ncbi.nih.gov. The scorescan also be calculated using the methods and algorithms of Crickmore etal. as described in the Background section, above.

The amino acid homology will be highest in critical regions of the toxinwhich account for biological activity or are involved in thedetermination of three-dimensional configuration which ultimately isresponsible for the biological activity. In this regard, certain aminoacid substitutions are acceptable and can be expected if thesesubstitutions are in regions which are not critical to activity or areconservative amino acid substitutions which do not affect thethree-dimensional configuration of the molecule. For example, aminoacids may be placed in the following classes: non-polar, unchargedpolar, basic, and acidic. Conservative substitutions whereby an aminoacid of one class is replaced with another amino acid of the same typefall within the scope of the subject invention so long as thesubstitution does not materially alter the biological activity of thecompound. Table 2 provides a listing of examples of amino acidsbelonging to each class.

TABLE 2 Class of Amino Acid Examples of Amino Acids Nonpolar Ala, Val,Leu, Ile, Pro, Met, Phe, Trp Uncharged Polar Gly, Ser, Thr, Cys, Tyr,Asn, Gln Acidic Asp, Glu Basic Lys, Arg, His

In some instances, non-conservative substitutions can also be made. Thecritical factor is that these substitutions must not significantlydetract from the biological activity of the toxin.

As used herein, reference to “isolated” polynucleotides and/or“purified” toxins refers to these molecules when they are not associatedwith the other molecules with which they would be found in nature; theseterms would include their use in plants. Thus, reference to “isolated”and/or “purified” signifies the involvement of the “hand of man” asdescribed herein.

Synthetic genes which are functionally equivalent to the toxins of thesubject invention can also be used to transform hosts. Methods for theproduction of synthetic genes can be found in, for example, U.S. Pat.No. 5,380,831.

Transgenic hosts. The toxin-encoding genes of the subject invention canbe introduced into a wide variety of microbial or plant hosts. Inpreferred embodiments, expression of the toxin gene results, directly orindirectly, in the intracellular production and maintenance of thepesticide proteins. When transgenic/recombinant/transformed host cellsare ingested by the pests, the pests will ingest the toxin. This is thepreferred manner in which to cause contact of the pest with the toxin.The result is a control (killing or making sick) of the pest.Alternatively, suitable microbial hosts, e.g.; Pseudomonas such as P.fluorescens, can be applied to the situs of the pest, where some ofwhich can proliferate, and are ingested by the target pests. The microbehosting the toxin gene can be treated under conditions that prolong theactivity of the toxin and stabilize the cell. The treated cell, whichretains the toxic activity, then can be applied to the environment ofthe target pest.

In preferred embodiments, recombinant plant cells and plants are used.Preferred plants (and plant cells) are corn and/or maize.

Where the B.t. toxin gene is introduced via a suitable vector into amicrobial host, and said host is applied to the environment in a livingstate, certain host microbes should be used. Microorganism hosts areselected which are known to occupy the “phytosphere” (phylloplane,phyllosphere, rhizosphere, and/or rhizoplane) of one or more crops ofinterest. These microorganisms are selected so as to be capable ofsuccessfully competing in the particular environment (crop and otherinsect habitats) with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the polypeptidepesticide, and, desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

A large number of microorganisms are known to inhabit the phylloplane(the surface of the plant leaves) and/or the rhizosphere (the soilsurrounding plant roots) of a wide variety of important crops. Thesemicroorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms, such as bacteria, e.g., genera Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylophilius, Agrobacterium, Acetobacter,Lactobacillus, Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes;fungi, particularly yeast, e.g., genera Saccharomyces, Cryptococcus,Kluyveromyces, Sporobolomyces, Rhodotorula, and Aureobasidium. Ofparticular interest are such phytosphere bacterial species asPseudomonas syringae, Pseudomonas fluorescens, Serratia marcescens,Acetobacter xylinum, Agrobacterium tumefaciens, Rhodopseudomonasspheroides, Xanthomonas campestris, Rhizobium melioti, Alcaligenesentrophus, and Azotobacter vinlandii; and phytosphere yeast species suchas Rhodotorula rubra, R. glutinis, R. marina, R. aurantiaca,Cryptococcus albidus, C. diffluens, C. laurentii, Saccharomyces rosei,S. pretoriensis, S. cerevisiae, Sporobolomyces roseus, S. odorus,Kluyveromyces veronae, and Aureobasidium pollulans. Of particularinterest are the pigmented microorganisms.

A wide variety of ways are available for introducing a B.t. geneencoding a toxin into the target host under conditions which allow forstable maintenance and expression of the gene. These methods are wellknown to those skilled in the art and are described, for example, inU.S. Pat. No. 5,135,867, which is incorporated herein by reference.

Treatment of cells. As mentioned above, B.t. or recombinant cellsexpressing a B.t. toxin can be treated to prolong the toxin activity andstabilize the cell. The pesticide microcapsule that is formed comprisesthe B.t. toxin within a cellular structure that has been stabilized andwill protect the toxin when the microcapsule is applied to theenvironment of the target pest. Suitable host cells may include eitherprokaryotes or eukaryotes, normally being limited to those cells whichdo not produce substances toxic to higher organisms, such as mammals.However, organisms which produce substances toxic to higher organismscould be used, where the toxic substances are unstable or the level ofapplication sufficiently low as to avoid any possibility of toxicity toa mammalian host. As hosts, of particular interest will be theprokaryotes and the lower eukaryotes, such as fungi.

The cell will usually be intact and be substantially in theproliferative form when treated, rather than in a spore form, althoughin some instances spores may be employed.

Treatment of the microbial cell, e.g., a microbe containing the B.t.toxin gene, can be by chemical or physical means, or by a combination ofchemical and/or physical means, so long as the technique does notdeleteriously affect the properties of the toxin, nor diminish thecellular capability of protecting the toxin. Examples of chemicalreagents are halogenating agents, particularly halogens of atomic no.17-80. More particularly, iodine can be used under mild conditions andfor sufficient time to achieve the desired results. Other suitabletechniques include treatment with aldehydes, such as glutaraldehyde;anti-infectives, such as zephiran chloride and cetylpyridinium chloride;alcohols, such as isopropyl and ethanol; various histologic fixatives,such as Lugol iodine, Bouin's fixative, various acids and Helly'sfixative (See: Humason, Gretchen L., Animal Tissue Techniques, W. H.Freeman and Company, 1967); or a combination of physical (heat) andchemical agents that preserve and prolong the activity of the toxinproduced in the cell when the cell is administered to the hostenvironment. Examples of physical means are short wavelength radiationsuch as gamma-radiation and X-radiation, freezing, UV irradiation,lyophilization, and the like. Methods for treatment of microbial cellsare disclosed in U.S. Pat. Nos. 4,695,455 and 4,695,462, which areincorporated herein by reference.

The cells generally will have enhanced structural stability which willenhance resistance to environmental conditions. Where the pesticide isin a proform, the method of cell treatment should be selected so as notto inhibit processing of the proform to the mature form of the pesticideby the target pest pathogen. For example, formaldehyde will crosslinkproteins and could inhibit processing of the proform of a polypeptidepesticide. The method of treatment should retain at least a substantialportion of the bio-availability or bioactivity of the toxin.

Characteristics of particular interest in selecting a host cell forpurposes of production include ease of introducing the B.t. gene intothe host, availability of expression systems, efficiency of expression,stability of the pesticide in the host, and the presence of auxiliarygenetic capabilities. Characteristics of interest for use as a pesticidemicrocapsule include protective qualities for the pesticide, such asthick cell walls, pigmentation, and intracellular packaging or formationof inclusion bodies; survival in aqueous environments; lack of mammaliantoxicity; attractiveness to pests for ingestion; ease of killing andfixing without damage to the toxin; and the like. Other considerationsinclude ease of formulation and handling, economics, storage stability,and the like.

Growth of cells. The cellular host containing the B.t. insecticidal genemay be grown in any convenient nutrient medium, preferably where the DNAconstruct provides a selective advantage, providing for a selectivemedium so that substantially all or all of the cells retain the B.t.gene. These cells may then be harvested in accordance with conventionalways. Alternatively, the cells can be treated prior to harvesting.

The B.t. cells of the invention can be cultured using standard art mediaand fermentation techniques. Upon completion of the fermentation cyclethe bacteria can be harvested by first separating the B.t. spores andcrystals from the fermentation broth by means well known in the art. Therecovered B.t. spores and crystals can be formulated into a wettablepowder, liquid concentrate, granules or other formulations by theaddition of surfactants, dispersants, inert carriers, and othercomponents to facilitate handling and application for particular targetpests. These formulations and application procedures are all well knownin the art.

Formulations. Formulated bait granules containing an attractant andspores and crystals of the B.t. isolates, or recombinant microbescomprising the genes obtainable from the B.t. isolates disclosed herein,can be applied to the soil. Formulated product can also be applied as aseed-coating or root treatment or total plant treatment at later stagesof the crop cycle. Plant and soil treatments of B.t. cells may beemployed as wettable powders, granules or dusts, by mixing with variousinert materials, such as inorganic minerals (phyllosilicates,carbonates, sulfates, phosphates, and the like) or botanical materials(powdered corncobs, rice hulls, walnut shells, and the like). Theformulations may include spreader-sticker adjuvants, stabilizing agents,other pesticidal additives, or surfactants. Liquid formulations may beaqueous-based or non-aqueous and employed as foams, gels, suspensions,emulsifiable concentrates, or the like. The ingredients may includerheological agents, surfactants, emulsifiers, dispersants, or polymers.

As would be appreciated by a person skilled in the art, the pesticidalconcentration will vary widely depending upon the nature of theparticular formulation, particularly whether it is a concentrate or tobe used directly. The pesticide will be present in at least 1% by weightand may be 100% by weight. The dry formulations will have from about1-95% by weight of the pesticide while the liquid formulations willgenerally be from about 1-60% by weight of the solids in the liquidphase. The formulations will generally have from about 10² to about 10⁴cells/mg. These formulations will be administered at about 50 mg (liquidor dry) to 1 kg or more per hectare.

The formulations can be applied to the environment of the pest, e.g.,soil and foliage, by spraying, dusting, sprinkling, or the like.

Mutants. Mutants of the isolates of the invention can be made byprocedures well known in the art. For example, an asporogenous mutantcan be obtained through ethylmethane sulfonate (EMS) mutagenesis of anisolate. The mutants can be made using ultraviolet light andnitrosoguanidine by procedures well known in the art.

A smaller percentage of the asporogenous mutants will remain intact andnot lyse for extended fermentation periods; these strains are designatedlysis minus (−). Lysis minus strains can be identified by screeningasporogenous mutants in shake flask media and selecting those mutantsthat are still intact and contain toxin crystals at the end of thefermentation. Lysis minus strains are suitable for a cell treatmentprocess that will yield a protected, encapsulated toxin protein.

To prepare a phage resistant variant of said asporogenous mutant, analiquot of the phage lysate is spread onto nutrient agar and allowed todry. An aliquot of the phage sensitive bacterial strain is then plateddirectly over the dried lysate and allowed to dry. The plates areincubated at 30° C. The plates are incubated for 2 days and, at thattime, numerous colonies could be seen growing on the agar. Some of thesecolonies are picked and subcultured onto nutrient agar plates. Theseapparent resistant cultures are tested for resistance by cross streakingwith the phage lysate. A line of the phage lysate is streaked on theplate and allowed to dry. The presumptive resistant cultures are thenstreaked across the phage line. Resistant bacterial cultures show nolysis anywhere in the streak across the phage line after overnightincubation at 30° C. The resistance to phage is then reconfirmed byplating a lawn of the resistant culture onto a nutrient agar plate. Thesensitive strain is also plated in the same manner to serve as thepositive control. After drying, a drop of the phage lysate is placed inthe center of the plate and allowed to dry. Resistant cultures showed nolysis in the area where the phage lysate has been placed afterincubation at 30° C. for 24 hours.

Following are examples which illustrate procedures for practicing theinvention. These examples should not be construed as limiting. Allpercentages are by weight and all solvent mixture proportions are byvolume unless otherwise noted.

EXAMPLE 1 Culturing of B.t. Isolates of the Invention

A subculture of the B.t. isolates, or mutants thereof, can be used toinoculate the following medium, a peptone, glucose, salts medium.

Bacto Peptone 7.5 g/l Glucose 1.0 g/l KH₂PO₄ 3.4 g/l K₂HPO₄ 4.35 g/lSalt Solution 5.0 ml/l CaCl₂ Solution 5.0 ml/l pH 7.2 Salts Solution(100 ml) MgSO₄.7 H₂O 2.46 g MnSO₄.H₂O 0.04 g ZnSO₄.7 H₂O 0.28 g FeSO₄.7H₂O 0.40 g CaCl₂ Solution (100 ml) CaCl₂.2 H₂O 3.66 g

The salts solution and CaCl₂ solution are filter-sterilized and added tothe autoclaved and cooked broth at the time of inoculation. Flasks areincubated at 30° C. on a rotary shaker at 200 rpm for 64 hr.

The above procedure can be readily scaled up to large fermentors byprocedures well known in the art.

The B.t. spores and/or crystals, obtained in the above fermentation, canbe isolated by procedures well known in the art. A frequently-usedprocedure is to subject the harvested fermentation broth to separationtechniques, e.g., centrifugation.

EXAMPLE 2 Activity of Sporulated Bacillus thuringiensis Cultures on CornRootworm

Liquid cultures of PS80JJ1, PS149B1 or PS167H2 were grown to sporulationin shake flasks and pelleted by centrifugation. Culture pellets wereresuspended in water and assayed for activity against corn rootworm intop load bioassays as described above. The amounts of 14 kDa and 44.3kDa proteins present in the culture pellets were estimated bydensitometry and used to calculate specific activity expressed as LC₅₀.Activity of each native B. thuringiensis strain is presented in Table 3(WCRW top load bioassay of B.t. strains).

TABLE 3 WCRW Top Load Bioassay of B.t. Strains B.t. strain LC₅₀(μg/cm²)* 95% CL Slope PS80JJ1 6  4-8 1.5 PS167H2 6  4-9 1.6 PS149B1 8  4-12 1.8 CryB cell blank 4% N/A N/A Water blank 4% N/A N/A *Percentagemortality at top dose is provided for controls

EXAMPLE 3 Protein Purification for 45 kDa 80JJ1 Protein

One gram of lyophilized powder of 80JJ1 was suspended in 40 ml of buffercontaining 80 mM Tris-Cl pH 7.8, 5 mM EDTA, 100 μM PMSF, 0.5 μg/mlLeupeptin, 0.7 μg/ml Pepstatin, and 40 μg/ml Bestatin. The suspensionwas centrifuged, and the resulting supernatant was discarded. The pelletwas washed five times using 35-40 ml of the above buffer for each wash.The washed pellet was resuspended in 10 ml of 40% NaBr, 5 mM EDTA, 100μM PMSF, 0.5 μg/ml Leupeptin, 0.7 μg/ml Pepstatin, and 40 μg/ml Bestatinand placed on a rocker platform for 75 minutes. The NaBr suspension wascentrifuged, the supernatant was removed, and the pellet was treated asecond time with 40% NaBr, 5 mM EDTA, 100 μM PMSF, 0.5 μg/ml Leupeptin,0.7 μg/ml Pepstatin, and 40 μg/ml Bestatin as above. The supernatants(40% NaBr soluble) were combined and dialyzed against 10 mM CAPS pH10.0, 1 mM EDTA at 4° C. The dialyzed extracts were centrifuged and theresulting supernatant was removed. The pellet (40% NaBr dialysis pellet)was suspended in 5 ml of H₂O and centrifuged. The resultant supernatantwas removed and discarded. The washed pellet was washed a second time in10 ml of H₂O and centrifuged as above. The washed pellet was suspendedin 1.5 ml of H₂O and contained primarily three protein bands withapparent mobilities of approximately 47 kDa, 45 kDa, and 15 kDa whenanalyzed using SDS-PAGE. At this stage of purification, the suspended40% NaBr dialysis pellet contained approximately 21 mg/ml of protein byLowry assay.

The proteins in the pellet suspension were separated using SDS-PAGE(Laemlli, U. K. [1970] Nature 227:680) in 15% acrylamide gels. Theseparated proteins were then electrophoretically blotted to a PVDFmembrane (Millipore Corp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 Vconstant. After one hour the PVDF membrane was rinsed in water brieflyand placed for 3 minutes in 0.25% Coomassie blue R-250, 50% methanol, 5%acetic acid. The stained membrane was destained in 40% MeOH, 5% aceticacid. The destained membrane was air-dried at room temperature (LeGendreet al. [1989] In A Practical Guide to Protein Purification ForMicrosequencing, P. Matsudaira, ed., Academic Press, New York, N.Y.).The membrane was sequenced using automated gas phase Edman degradation(Hunkapillar, M. W., R. M. Hewick, W. L. Dreyer, L. E. Hood [1983] Meth.Enzymol. 91:399).

The amino acid analysis revealed that the N-terminal sequence of the 45kDa band was as follows: Met-Leu-Asp-Thr-Asn (SEQ ID NO:1).

The 47 kDa band was also analyzed and the N-terminal amino acid sequencewas determined to be the same 5-amino acid sequence as SEQ ID NO:1.Therefore, the N-terminal amino acid sequences of the 47 kDa peptide andthe 45 kDa peptide were identical.

This amino acid sequence also corresponds to a sequence obtained from a45 kDa peptide obtained from PS80JJ1 spore/crystal powders, usinganother purification protocol, with the N-terminal sequence as follows:Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-Ile-Ser-Asn-Leu-Ala-Asn-Gly-Leu-Tyr-Thr-Ser-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO:2).

EXAMPLE 4 Purification of the 14 kDa Peptide of PS80JJ1

0.8 ml of the white dialysis suspension (approximately 21 mg/ml)containing the 47 kDa, 45 kDa, and 15 kDa peptides, was dissolved in 10ml of 40% NaBr, and 0.5 ml of 100 mM EDTA were added. After about 18hours (overnight), a white opaque suspension was obtained. This wascollected by centrifugation and discarded. The supernatant wasconcentrated in a Centricon-30 (Amicon Corporation) to a final volume ofapproximately 15 ml. The filtered volume was washed with water by filterdialysis and incubated on ice, eventually forming a milky whitesuspension. The suspension was centrifuged and the pellet andsupernatant were separated and retained. The pellet was then suspendedin 1.0 ml water (approximately 6 mg/ml). The pellet containedsubstantially pure 15 kDa protein when analyzed by SDS-PAGE.

The N-terminal amino acid sequence was determined to be:Ser-Ala-Arg-Glu-Val-His-Ile-Glu-Ile-Asn-Asn-Thr-Arg-His-Thr-Leu-Gln-Leu-Glu-Ala-Lys-Thr-Lys-Leu(SEQ ID NO:3).

EXAMPLE 5 Bioassay of Protein

A preparation of the insoluble fraction from the dialyzed NaBr extractof 80JJ1 containing the 47 kDa, 45 kDa, and 15 kDa peptides wasbioassayed against Western corn rootworm and were found to exhibitsignificant toxin activity.

EXAMPLE 6 Protein Purification and Characterization of PS149B1 45 kDaProtein

The P1 pellet was resuspended with two volumes of deionized water perunit wet weight, and to this was added nine volumes of 40% (w/w) aqueoussodium bromide. This and all subsequent operations were carried out onice or at 4-6° C. After 30 minutes, the suspension was diluted with 36volumes of chilled water and centrifuged at 25,000×g for 30 minutes togive a pellet and a supernatant.

The resulting pellet was resuspended in 1-2 volumes of water and layeredon a 20-40% (w/w) sodium bromide gradient and centrifuged at 8,000×g for100 minutes. The layer banding at approximately 32% (w/w) sodium bromide(the “inclusions”, or INC) was recovered and dialyzed overnight againstwater using a dialysis membrane with a 6-8 kDa MW cut-off. Particulatematerial was recovered by centrifugation at 25,000×g, resuspended inwater, and aliquoted and assayed for protein by the method of Lowry andby SDS-PAGE.

The resulting supernatant was concentrated 3- to 4-fold usingCentricon-10 concentrators, then dialyzed overnight against water usinga dialysis membrane with a 6-8 kDa MW cut-off. Particulate material wasrecovered by centrifugation at 25,000×g, resuspended in water, andaliquoted and assayed for protein by the method of Lowry and bySDS-PAGE. This fraction was denoted as P1.P2.

The peptides in the pellet suspension were separated using SDS-PAGE(Laemlli, U. K., supra) in 15% acrylamide gels. The separated proteinswere then electrophoretically blotted to a PVDF membrane (MilliporeCorp.) in 10 mM CAPS pH 11.0, 10% MeOH at 100 V constant. After one hourthe PVDF membrane was rinsed in water briefly and placed for 3 minutesin 0.25% Coomassie blue R-250, 50% methanol, 5% acetic acid. The stainedmembrane was destained in 40% MeOH, 5% acetic acid. The destainedmembrane was air-dried at room temperature (LeGendre et al., supra). Themembrane was sequenced using automated gas phase Edman degradation(Hunkapillar et al., supra).

Protein analysis indicated the presence of two major polypeptides, withmolecular weights of 47 kDa and 14 kDa. Molecular weights were measuredagainst standard polypeptides of known molecular weight. This processprovides only an estimate of true molecular weight. The 47 kDa band fromPS149B1 migrated on SDS-PAGE in a manner indistinguishable from the 47kDa protein from PS80JJ1. Likewise, the 14 kDa band from PS149B1migrated on SDS-PAGE in a manner indistinguishable from 14 kDa bandsfrom PS167H2 and PS80JJ1. Apart from these two polypeptides, which wereestimated to account for 25-35% (47 kDa) and 35-55% (15 kDa) of theCoomassie staining material respectively, there may be minor bands,including those of estimated MW at 46 kDa, 130 kDa, and 70 kDa.

Protein analysis indicated that fraction INC contained a singlepolypeptide with MW of 47 kDa, and that fraction P1.P2 contained asingle polypeptide with MW of 14 kDa. These polypeptides were recoveredin yields greater than 50% from P1.

The N-terminal amino acid sequence for the purified 47 kDa protein fromPS149B1 is:Met-Leu-Asp-Thr-Asn-Lys-Val-Tyr-Glu-Ile-Ser-Asn-His-Ala-Asn-Gly-Leu-Tyr-Ala-Ala-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO:4).

The N-terminal amino acid sequence for the purified 14 kDa protein fromPS149B1 is:Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp-Lys-Thr-Lys-Leu-Asp-Gly-Gly-Arg-Trp-Arg-Thr-Ser-Pro-Xaa-Asn-Val-Ala-Asn-Asp-Gln-Ile-Lys-Thr-Phe-Val-Ala-Glu-Ser-Asn (SEQID NO:5).

EXAMPLE 7 Amino Acid Sequence for 45 kDa and 14 kDa Toxins of PS167H2

The N-terminal amino acid sequence for the purified 45 kDa protein fromPS167H2 is:Met-Leu-Asp-Thr-Asn-Lys-Ile-Tyr-Glu-Ile-Ser-Asn-Tyr-Ala-Asn-Gly-Leu-His-Ala-Ala-Thr-Tyr-Leu-Ser-Leu(SEQ ID NO:6).

The N-terminal amino acid sequence for the purified 14 kDa protein fromPS167H2 is:Ser-Ala-Arg-Glu-Val-His-Ile-Asp-Val-Asn-Asn-Lys-Thr-Gly-His-Thr-Leu-Gln-Leu-Glu-Asp-Lys-Thr-Lys-Leu(SEQ ID NO:7).

These amino acid sequences can be compared to the sequence obtained forthe 47 kDa peptide obtained from 80JJ1 spore/crystal powders with theN-terminal sequence (SEQ ID NO:1) and to the sequence obtained for the14 kDa peptide obtained from 80JJ1 spore/crystal powders with theN-terminal sequence (SEQ ID NO:3).

Clearly, the 45-47 kDa proteins are highly related, and the 14 kDaproteins are highly related.

EXAMPLE 8 Bioassay of Protein

The purified protein fractions from PS149B1 were bioassayed againstwestern corn rootworm and found to exhibit significant toxin activitywhen combined. In fact, the combination restored activity to that notedin the original preparation (P1). The following bioassay data setpresents percent mortality and demonstrates this effect.

TABLE 4 Concentration (μg/cm²) PI INC P1.P2 INC + P1.P2 300 88, 100, 9419 13 100 100 94, 50, 63 31 38 94 33.3 19, 19, 44 38 13 50 11.1 13, 56,25 12 31 13 3.7 0, 50, 0 0 31 13 1.2 13, 43, 12 0 12 19 0.4 6, 12, 6 2519 6

EXAMPLE 9 Molecular Cloning, Expression, and DNA Sequence Analysis of aNovel δ-Endotoxin Gene From Bacillus thuringiensis Strain PS80JJ1

Total cellular DNA was prepared from Bacillus thuringiensis (B.t.) cellsgrown to an optical density, at 600 nm, of 1.0. Cells were pelleted bycentrifugation and resuspended in protoplast buffer (20 mg/ml lysozymein 0.3 M sucrose, 25 mM Tris-Cl [pH 8.0], 25 mM EDTA). After incubationat 37° C. for 1 hour, protoplasts were lysed by two cycles of freezingand thawing. Nine volumes of a solution of 0.1 M NaCl, 0.1% SDS, 0.1 MTris-Cl were added to complete lysis. The cleared lysate was extractedtwice with phenol:chloroform (1:1). Nucleic acids were precipitated withtwo volumes of ethanol and pelleted by centrifugation. The pellet wasresuspended in TE buffer and RNase was added to a final concentration of50 μg/ml. After incubation at 37° C. for 1 hour, the solution wasextracted once each with phenol:chloroform (1:1) and TE-saturatedchloroform. DNA was precipitated from the aqueous phase by the additionof one-tenth volume of 3 M NaOAc and two volumes of ethanol. DNA waspelleted by centrifugation, washed with 70% ethanol, dried, andresuspended in TE buffer.

An oligonucleotide probe for the gene encoding the PS80JJ1 45 kDa toxinwas designed from N-terminal peptide sequence data. The sequence of the29-base oligonucleotide probe was:

5′-ATG YTW GAT ACW AAT AAA GTW TAT GAA AT-3′ (SEQ ID NO:8)

This oligonucleotide was mixed at four positions as shown. This probewas radiolabeled with ³²P and used in standard condition hybridizationof Southern blots of PS80JJ1 total cellular DNA digested with variousrestriction endonucleases. Representative autoradiographic data fromthese experiments showing the sizes of DNA restriction fragmentscontaining sequence homology to the 44.3 kDa toxin oligonucleotide probeof SEQ ID NO:8 are presented in Table 5.

TABLE 5 RFLP of PS80JJ1 cellular DNA fragments on Southern blots thathybridized under standard conditions with the 44.3 kDa toxin geneoligonucleotide probe (SEQ ID NO: 8) Restriction Enzyme ApproximateFragment Size (kbp) EcoRI 6.0 HindIII 8.3 KpnI 7.4 PstI 11.5 XbaI 9.1

These DNA fragments identified in these analyses contain all or asegment of the PS80JJ1 45 kDa toxin gene. The approximate sizes of thehybridizing DNA fragments in Table 5 are in reasonable agreement withthe sizes of a subset of the PS80JJ1 fragments hybridizing with aPS80JJ1 45 kDa toxin subgene probe used in separate experiments, aspredicted (see Table 6, below).

A gene library was constructed from PS80JJ1 DNA partially digested withSau3AI. Partial restriction digests were fractionated by agarose gelelectrophoresis. DNA fragments 9.3 to 23 kbp in size were excised fromthe gel, electroeluted from the gel slice, purified on an Elutip-D ionexchange column (Schleicher and Schuell, Keene, N H), and recovered byethanol precipitation. The Sau3AI inserts were ligated intoBamHI-digested LambdaGem-11 (Promega, Madison, Wis.). Recombinant phagewere packaged and plated on E. coli KW251 cells. Plaques were screenedby hybridization with the oligonucleotide probe described above.Hybridizing phage were plaque-purified and used to infect liquidcultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

Southern blot analysis revealed that one of the recombinant phageisolates contained an approximately 4.8 kbp Xbal-SacI band thathybridized to the PS80JJ1 toxin gene probe. The SacI site flanking thePS80JJ1 toxin gene is a phage vector cloning site, while the flankingXbaI site is located within the PS80JJ1 DNA insert. This DNA restrictionfragment was subcloned by standard methods into pBluescript S/K(Stratagene, San Diego, Calif.) for sequence analysis. The resultantplasmid was designated pMYC2421. The DNA insert was also subcloned intopHTBlueII (an E. coli/B. thuringiensis shuttle vector comprised ofpBluescript S/K [Stratagene, La Jolla, Calif.] and the replicationorigin from a resident B.t. plasmid [D. Lereclus et al. (1989) FEMSMicrobiology Letters 60:211-218]) to yield pMYC2420.

An oligonucleotide probe for the gene encoding the PS80JJ1 14 kDa toxinwas designed from N-terminal peptide sequence data. The sequence of the28-base oligonucleotide probe was: 5′ GW GAA GTW CAT ATW GAA ATW AAT AATAC 3′ (SEQ ID NO:29). This oligonucleotide was mixed at four positionsas shown. The probe was radiolabelled with ³²P and used in standardcondition hybridizations of Southern blots of PS80JJ1 total cellular andpMYC2421 DNA digested with various restriction endonucleases. These RFLPmapping experiments demonstrated that the gene encoding the 14 kDa toxinis located on the same genomic EcoRI fragment that contains theN-terminal coding sequence for the 44.3 kDa toxin.

To test expression of the PS80JJ1 toxin genes in B.t., pMYC2420 wastransformed into the acrystalliferous (Cry-) B.t. host, CryB (A.Aronson, Purdue University, West Lafayette, Ind.), by electroporation.Expression of both the approximately 14 and 44.3 kDa PS80JJ1 toxinsencoded by pMYC2420 was demonstrated by SDS-PAGE analysis. Toxin crystalpreparations from the recombinant CryB[pMYC2420] strain, MR536, wereassayed and found to be active against western corn rootworm.

The PS80JJ1 toxin genes encoded by pMYC2421 were sequenced using theABI373 automated sequencing system and associated software. The sequenceof the entire genetic locus containing both open reading frames andflanking nucleotide sequences is shown in SEQ ID NO:30. The terminationcodon of the 14 kDa toxin gene is 121 base pairs upstream (5′) from theinitiation codon of the 44.3 kDa toxin gene (FIG. 2). The PS80JJ1 14 kDatoxin open reading frame nucleotide sequence (SEQ ID NO:31), the 44.3kDa toxin open reading frame nucleotide sequence (SEQ ID NO:10), and therespective deduced amino acid sequences (SEQ ID NO:32 and SEQ ID NO:11)are novel compared to other toxin genes encoding pesticidal proteins.

Thus, the nucleotide sequence encoding the 14 kDa toxin of PS80JJ1 isshown in SEQ ID NO:31. The deduced amino acid sequence of the 14 kDatoxin of PS80JJ1 is shown in SEQ ID NO:32. The nucleotide sequencesencoding both the 14 and 45 kDa toxins of PS80JJ1, as well as theflanking sequences, are shown in SEQ ID NO:30. The relationship of thesesequences is shown in FIG. 2.

A subculture of E. coli NM522 containing plasmid pMYC2421 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 28, 1996. The accession number is NRRL B-21555.

EXAMPLE 10 RFLP and PCR Analysis of Additional Novel δ-Endotoxin GenesFrom Bacillus thuringiensis Strains PS149B1 and PS167H2

Two additional strains active against corn rootworm, PS149B1 andPS167H2, also produce parasporal protein crystals comprised in part ofpolypeptides approximately 14 and 45 kDa in size. Southern hybridizationand partial DNA sequence analysis were used to examine the relatednessof these toxins to the 80JJ1 toxins. DNA was extracted from these B.t.strains as described above, and standard Southern hybridizations wereperformed using the 14 kDa toxin oligonucleotide probe (SEQ ID NO:29)and an approximately 800 bp PCR fragment of the 80JJ1 44.3 kDa toxingene-encoding sequence. RFLP data from these experiments showing thesizes of DNA restriction fragments containing sequence homology to the44.3 kDa toxin are presented in Table 6. RFLP data from theseexperiments showing the sizes of DNA restriction fragments containingsequence homology to the approximately 14 kDa toxin are presented inTable 7.

TABLE 6 RFLP of PS80JJ1, PS149B1, and PS167H2 cellular DNA fragments onSouthern blots that hybridized with the approximately 800 bp PS80JJ144.3 kDa toxin subgene probe under standard conditions Strain PS80JJ1PS149B1 PS167H2 Restriction enzyme Approximate fragment size (kbp) EcoRI6.4 5.7 2.6 1.3 2.8 0.6 HindIII 8.2 6.2 4.4 KpnI 7.8 10.0 11.5 PstI 12.09.2 9.2 8.2 XbaI 9.4 10.9 10.9 SacI 17.5 15.5 11.1 13.1 10.5 6.3

Each of the three strains exhibited unique RFLP patterns. Thehybridizing DNA fragments from PS149B1 or PS167H2 contain all or part oftoxin genes with sequence homology to the PS80JJ1 44.3 kDa toxin.

TABLE 7 Restriction fragment length polymorphisms of PS80JJ1, PS149B1,and PS167H2 cellular DNA fragments on Southern blots that hybridizedwith the PS80JJ1 14 kDa toxin oligonucleotide probe under standardconditions Strain PS80JJ1 PS149B1 PS167H2 Restriction enzyme Approximatefragment size (kbp) EcoRI 5.6 2.7 2.7 HindIII 7.1 6.0 4.7 XbaI 8.4 11.211.2

Each of the three strains exhibited unique RFLP patterns. Thehybridizing DNA fragments from PS149B1 or PS167H2 contain all or part oftoxin genes with sequence homology to the PS80JJ1 14 kDa toxin gene.

Portions of the toxin genes in PS149B1 or PS167H2 were amplified by PCRusing forward and reverse oligonucleotide primer pairs designed based onthe PS80JJ1 44.3 kDa toxin gene sequence. For PS149B1, the followingprimer pair was used:

Forward:

5′-ATG YTW GAT ACW AAT AAA GTW TAT GAA AT-3′ (SEQ ID NO:8)

Reverse:

5′-GGA TTA TCT ATC TCT GAG TGT TCT TG-3′ (SEQ ID NO:9)

For PS167H2, the same primer pair was used. These PCR-derived fragmentswere sequenced using the ABI373 automated sequencing system andassociated software. The partial gene and peptide sequences obtained areshown in SEQ ID NO:12-15. These sequences contain portions of thenucleotide coding sequences and peptide sequences for novel cornrootworm-active toxins present in B.t. strains PS149B1 or PS167H2.

EXAMPLE 11 Molecular Cloning and DNA Sequence Analysis of Novelδ-Endotoxin Genes From Bacillus thuringiensis Strains PS149B1 andPS167H2

Total cellular DNA was extracted from strains PS149B1 and PS167H2 asdescribed for PS80JJ1. Gene libraries of size-fractionated Sau3A partialrestriction fragments were constructed in Lambda-Gem11 for eachrespective strain as previously described. Recombinant phage werepackaged and plated on E. coli KW251 cells. Plaques were screened byhybridization with the oligonucleotide probe specific for the 44 kDatoxin gene. Hybridizing phage were plaque-purified and used to infectliquid cultures of E. coli KW251 cells for isolation of DNA by standardprocedures (Maniatis et al., supra).

For PS167H2, Southern blot analysis revealed that one of the recombinantphage isolates contained an approximately 4.0 to 4.4 kbp HindIII bandthat hybridized to the PS80JJ1 44 kDa toxin gene 5′ oligonucleotideprobe (SEQ ID NO:8). This DNA restriction fragment was subcloned bystandard methods into pBluescript S/K (Stratgene, San Diego, Calif.) forsequence analysis. The fragment was also subcloned into the high copynumber shuttle vector, pHT370 (Arantes, O., D. Lereclus [1991] Gene108:115-119) for expression analyses in Bacillus thuringiensis (seebelow). The resultant recombinant, high copy number bifunctional plasmidwas designated pMYC2427.

The PS167H2 toxin genes encoded by pMYC2427 were sequenced using the ABIautomated sequencing system and associated software. The sequence of theentire genetic locus containing both open reading frames and flankingnucleotide sequences is shown in SEQ ID NO:34. The termination codon ofthe 14 kDa toxin gene is 107 base pairs upstream (5′) from theinitiation codon of the 44 kDa toxin gene. The PS167H2 14 kDa toxincoding sequence (SEQ ID NO:35), the 44 kDa toxin coding sequence (SEQ IDNO:37), and the respective deduced amino acid sequences, SEQ ID NO:36and SEQ ID NO:38, are novel compared to other known toxin genes encodingpesticidal proteins. The toxin genes are arranged in a similar mannerto, and have some homology with, the PS80JJ1 14 and 44 kDa toxins.

A subculture of E. coli NM522 containing plasmid pMYC2427 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 26, 1997. The accession number is NRRL B-21672.

For PS149B1, Southern blot analysis using the PS80JJ1 44 kDaoligonucleotide 5′ probe (SEQ ID NO:8) demonstrated hybridization of anapproximately 5.9 kbp ClaI DNA fragment. Complete ClaI digests ofPS149B1 genomic DNA were size fractionated on agarose gels and clonedinto pHTBlueII. The fragment was also subcloned into the high copynumber shuttle vector, pHT370 (Arantes, O., D. Lereclus [1991] Gene108:115-119) for expression analyses in Bacillus thuringiensis (seebelow). The resultant recombinant, high copy number bifunctional plasmidwas designated pMYC2429.

The PS149B1 toxin genes encoded by pMYC2429 were sequenced using the ABIautomated sequencing system and associated software. The sequence of theentire genetic locus containing both open reading frames and flankingnucleotide sequences is shown in SEQ ID NO:39. The termination codon ofthe 14 kDa toxin gene is 108 base pairs upstream (5′) from theinitiation codon of the 44 kDa toxin gene. The PS149B1 14 kDa toxincoding sequence (SEQ ID NO:40), the 44 kDa toxin coding sequence (SEQ IDNO:42), and the respective deduced amino acid sequences, SEQ ID NO:41and SEQ ID NO:43, are novel compared to other known toxin genes encodingpesticidal proteins. The toxin genes are arranged in a similar manneras, and have some homology with, the PS80JJ1 and PS167H2 14 and 44 kDatoxins. Together, these three toxin operons comprise a new family ofpesticidal toxins.

A subculture of E. coli NM522 containing plasmid pMYC2429 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 26, 1997. The accession number is NRRL B-21673.

EXAMPLE 12 PCR Amplification for Identification and Cloning Novel CornRootworm-active Toxin

The DNA and peptide sequences of the three novel approximately 45 kDacorn rootworm-active toxins from PS80JJ1, PS149B1, and PS167H2 (SEQ IDNOS. 12-15) were aligned with the Genetics Computer Group sequenceanalysis program Pileup using a gap weight of 3.00 and a gap lengthweight of 0.10. The sequence alignments were used to identify conservedpeptide sequences to which oligonucleotide primers were designed thatwere likely to hybridize to genes encoding members of this novel toxinfamily. Such primers can be used in PCR to amplify diagnostic DNAfragments for these and related toxin genes. Numerous primer designs tovarious sequences are possible, four of which are described here toprovide an example. These peptide sequences are:

Asp-Ile-Asp-Asp-Tyr-Asn-Leu (SEQ ID NO:16)

Trp-Phe-Leu-Phe-Pro-Ile-Asp (SEQ ID NO:17)

Gln-Ile-Lys-Thr-Thr-Pro-Tyr-Tyr (SEQ ID NO:18)

Tyr-Glu-Trp-Gly-Thr-Glu (SEQ ID NO:19).

The corresponding nucleotide sequences are:

5′-GATATWGATGAYTAYAAYTTR-3′ (SEQ ID NO:20)

5′-TGGTTTTTRTTTCCWATWGAY-3′ (SEQ ID NO:21)

5′-CAAATHAAAACWACWCCATATTAT-3′ (SEQ ID NO:22)

5′-TAYGARTGGGGHACAGAA-3′ (SEQ ID NO:23).

Forward primers for polymerase amplification in thermocycle reactionswere designed based on the nucleotide sequences of SEQ ID NOs:20 and 21.

Reverse primers were designed based on the reverse complement of SEQ IDNOs:22 and 23:

5′-ATAATATGGWGTWGTTTTDATTTG-3′ (SEQ ID NO:24)

5′-TTCTGTDCCCCAYTCRTA-3′ (SEQ ID NO:25).

These primers can be used in combination to amplify DNA fragments of thefollowing sizes (Table 8) that identify genes encoding novel cornrootworm toxins.

TABLE 8 Predicted sizes of diagnostic DNA fragments (base pairs)amplifiable with primers specific for novel corn rootworm-active toxinsPrimer pair (SEQ ID NO.) DNA fragment size (bp) 20 + 24 495 20 + 25 59421 + 24 471 21 + 25 580

Similarly, entire genes encoding novel corn rootworm-active toxins canbe isolated by polymerase amplification in thermocycle reactions usingprimers designed based on DNA sequences flanking the open readingframes. For the PS80JJ1 44.3 kDa toxin, one such primer pair wasdesigned, synthesized, and used to amplify a diagnostic 1613 bp DNAfragment that included the entire toxin coding sequence. These primersare:

Forward: 5′-CTCAAAGCGGATCAGGAG-3′ (SEQ ID NO:26)

Reverse: 5′-GCGTATTCGGATATGCTTGG-3′ (SEQ ID NO:27).

For PCR amplification of the PS80JJ1 14 kDa toxin, the oligonucleotidecoding for the N-terminal peptide sequence (SEQ ID NO:29) can be used incombination with various reverse oligonucleotide primers based on thesequences in the PS80JJ1 toxin gene locus. One such reverse primer hasthe following sequence:

5′ CATGAGATTTATCTCCTGATCCGC 3′ (SEQ ID NO:33).

When used in standard PCR reactions, this primer pair amplified adiagnostic 1390 bp DNA fragment that includes the entire 14 kDa toxincoding sequence and some 3′ flanking sequences corresponding to the 121base intergenic spacer and a portion of the 44.3 kDa toxin gene. Whenused in combination with the 14 kDa forward primer, PCR will generate adiagnostic 322 base pair DNA fragment.

EXAMPLE 13 Clone Dose-response Bioassays

The PS80JJ1 toxin operon was subcloned from pMYC2421 into pHT370 fordirect comparison of bioactivity with the recombinant toxins cloned fromPS149B1 and PS167H2. The resultant recombinant, high copy numberbifunctional plasmid was designated pMYC2426.

A subculture of E. coli NM522 containing plasmid pMYC2426 was depositedin the permanent collection of the Patent Culture Collection (NRRL),Regional Research Center, 1815 North University Street, Peoria, Ill.61604 USA on Mar. 26, 1997. The accession number is NRRL B-21671.

To test expression of the PS80JJ1, PS149B1 and PS167H2 toxin genes inB.t., pMYC2426, pMYC2427 and pMYC2429 were separately transformed intothe acrystalliferous (Cry-) B.t. host, CryB (A. Aronson, PurdueUniversity, West Lafayette, Ind.), by electroporation. The recombinantstrains were designated MR543 (CryB [pMYC2426]), MR544 (CryB [pMYC2427])and MR546 (CryB [pMYC2429]), respectively. Expression of both theapproximately 14 and 44 kDa toxins was demonstrated by SDS-PAGE analysisfor each recombinant strain.

Toxin crystal preparations from the recombinant strains were assayedagainst western corn rootworm. Their diet was amended with sorbic acidand SIGMA pen-strep-ampho-B. The material was top-loaded at a rate of 50μl of suspension per cm² diet surface area. Bioassays were run withneonate Western corn rootworm larvae for 4 days at approximately 25° C.Percentage mortality and top-load LC₅₀ estimates for the clones(pellets) are set forth in Table 9. A dH2O control yielded 7% mortality.

TABLE 9 Percentage mortality at given protein concentration (μg/cm²)Sample 50 μg/cm² 5 μg/cm² 0.5 μg/cm² MR543 pellet 44% 19%  9% MR544pellet 72% 32% 21% MR546 pellet 52% 32% 21%

The amounts of 14 kDa and 44.3 kDa proteins present in the crystalpreparations were estimated by densitometry and used to calculatespecific activity expressed as LC₅₀. LC₅₀ estimates for the clones(pellets) are set forth in Table 10 (WCRW top load bioassay of B.t.clones).

TABLE 10 WCRW Top Load Bioassay of B.t. Clones B.t. Parental B.t. CloneStrain LC₅₀ (μg/cm²)* 95% CL Slope MR543 PS80JJ1 37  17-366* 0.79 MR544PS167H2 10  6-14  1.6 MR546 PS149B1 8 4-12  1.5 N/A CzyB cell blank  4%N/A N/A N/A Water blank  4% N/A N/A *Percentage mortality at top dose isprovided for controls **90% CL

EXAMPLE 14 Mutational Analysis of the 14 and 44 kDa Polypeptides in thePS80JJ1 Binary Toxin Operon

Binary toxin genes of the subject invention are, in their wild-typestate, typically arranged in an operon wherein the 14 kDa protein geneis transcribed first, followed by that of the 45 kDa protein gene. Thesegenes are separated by a relatively short, non-coding region.Representative ORFs are shown in SEQ ID NO:30, SEQ ID NO:34, and SEQ IDNO:39.

In order to investigate the contribution of the individual 14 and 44.3kDa crystal proteins to corn rootworm activity, each gene in the PS80JJ1operon was mutated in separate experiments to abolish expression of oneof the proteins. The intact gene was then expressed in B.t. andrecombinant proteins were tested for activity against corn rootworm.

First, the 44.3 kDa gene encoded on pMYC2421 was mutated by truncationat the EcoRI site at base position 387 of the open reading frame. Thistruncation and subsequent ligation with vector sequences resulted in anopen reading frame encoding an approximately 24 kDa hypothetical fusionprotein. The resulting operon encoding the intact 14 kDa gene and thetruncated 45 kDa gene was subcloned into the high copy number shuttlevector, pHT370 (Arantes, O., D. Lereclus [1991] Gene 108:115-119) forexpression analyses in Bacillus thuringiensis. The resulting plasmid,pMYC2424 was transformed into the acrystalliferous (Cry-) B.t. host,CryB (A. Aronson, Purdue University, West Lafayette, Ind.), byelectroporation. The resulting recombinant strain was designated MR541.Only the 14 kDa PS80JJ1 protein was detectable by SDSPAGE analysis ofsporulated cultures of MR541. Mortality was not observed forpreparations of MR541 expressing only the 14 kDa PS80JJ1 protein intop-load bioassays against corn rootworm.

Next, the 14 kDa gene encoded on pMYC2421 was mutated by insertion of anoligonucleotide linker containing termination codons in all possiblereading frames at the Nrul site at base position 11 of the open readingframe. The sequence of this linker is 5′ TGAGTAACTAGATCTATTCAATTA 3′.The linker introduces a BglII site for confirmation of insertion byBglII restriction digestion. Plasmid clones containing the mutageniclinker were identified with BglII and sequenced for verification. Theoperon insert encoding the 14 kDa nonsense mutations was subcloned intopHT370, resulting in plasmid pMYC2425. This plasmid was transformed intoCryB by electroporation to yield the recombinant B.t. strain MR542. Onlythe 44.3 kDa PS80JJ1 protein was expressed in sporulated cultures ofMR542 as shown by SDSPAGE analysis. Mortality against corn rootworm wasnot observed for preparations of MR542 expressing only the 44.3 kDaPS80JJ1 protein.

EXAMPLE 15 Single Gene Heterologous Expression, Purification andBioassay of the 14 and 44.3 kDa Polypeptides From PS149B1 in Pseudomonasfluorescens

The 14 kDa and 44.3 kDa polypeptide genes from PS149B1 were separatelyengineered into plasmid vectors by standard DNA cloning methods, andtransformed into Psuedomonas flourescens. The recombinant Pseudomonasfluorescens strain expressing only the PS149B1 14 kDa gene wasdesignated MR1253. The recombinant Pseudomonas fluorescens strainexpressing only the PS149B1 44.3 kDa gene was designated MR1256.

MR1253 and MR1256 each individually expressing one of the two binaryproteins were grown in 1 L fermentation tanks. A portion of each culturewas then pelleted by centrifugation, lysed with lysozyme, and treatedwith DNAse I to obtain semi-pure protein inclusions. These inclusionswere then solubilized in 50 mM Sodium Citrate (pH 3.3) by gentle rockingat 4° C. for 1 hour. The 14 kDa protein dissolved readily in this bufferwhereas the 44.3 kDa protein was partially soluble. The solubilizedfractions were then centrifuged at 15,000×g for 20 minutes; and thesupernatants were retained.

The 14 kDa protein was further purified through ion-exchangechromatography. The solubilized 14 kDa protein was bound to a Econo-Scolumn and eluted with a Sodium Chloride 0-1M gradient.

The chromatographically pure MR1253 (14 kDa protein) and the SodiumCitrate (pH3.3) solubilized preparation of MR1256 (45 kDa protein) werethen tested for activity on corn rootworm individually or together at amolar ratio of 1 to 10 (45 kDa protein to 14 kDa protein). Observedmortality for each of the proteins alone was not above background levels(of the water/control sample) but 87% mortality resulted when they werecombined in the above ratio (see Table 11).

TABLE 11 Molar ratio load ug 45 kD/ ug 14 kD/ Total ug CRW (45 kD to 14kD) volume well well protein Mortality 0 to 1 100 ul 0 260 260 13 1 to 0200 ul 260 0 260 9  1 to 10 100 ul 65 195 260 87 water 100 ul 0 0 0 11

EXAMPLE 16 Identification of Additional Novel 14 kDa and 44.3 kDa ToxinGenes by Hybridization of Total B.t. Genomic DNA and by RFLP

Total genomic DNA from each isolate was prepared using the Qiagen DNEasy96 well tissue kit. DNA in 96-well plates was denatured prior toblotting by adding 10 ul of each DNA sample and 10 ul of 4 M NaOH to 80ul sterile distilled water. Samples were incubated at 70° C. for onehour after which 100 ul of 20×SSC was added to each well. PS149B1 totalgenomic DNA was included with each set of 94 samples as a positivehybridization control, and cryb- total genomic DNA was included witheach set of 94 samples as a negative hybridization control. Each set of96 samples was applied to Magnacharge nylon membranes using two 48 wellslot blot manifolds (Hoefer Scientific), followed by two washes with10×SSC. Membranes were baked at 80° C. for one hour and kept dry untilused. Membranes were prehybridized and hybridized in standard formamidesolution (50% formamide, 5×SSPE, 5×Denhardt's solution, 2% SDS, 100ug/ml single stranded DNA) at 42° C. Membranes were washed under twoconditions: 2×SSC/0.1% SDS at 42° C. (low stringency) and 0.2×SSC/0.1%SDS at 65° C. (moderate to high stringency). Membranes were probed withan approximately 1.3 kilobase pair PCR fragment of the P5149B1 44.3 kDagene amplified from pMYC2429 using forward primer SEQ ID NO:8 and areverse primer with the sequence 5′ GTAGAAGCAGAACAAGAAGGTATT 3′ (SEQ IDNO:46). The probe was radioactively labeled using the Prime-it II kit(Stratagene) and 32-P-dCTP, purified on Sephadex columns, denatured at94° C. and added to fresh hybridization solution. Strains containinggenes with homology to the PS149B1 probe were identified by exposingmembranes to X-ray film.

The following strains were identified by positive hybridizationreactions: PS184M2, PS185GG, PS187G1, PS187Y2, PS201G, PS201HH2,PS242K10, PS69Q, KB54A1-6, KR136, KR589, PS185L12, PS185W3, PS185Z11,PS186L9, PS187L14, PS186FF, PS131W2, PS147U2, PS158T3, PS158X10,PS185FF, PS187F3, PS198H3, PS201H2, PS201L3, PS203G2, PS203J1, PS204C3,PS204G4, PS204I11, PS204J7, PS210B, PS213E8, PS223L2, PS224F2, PS236B6,PS246P42, PS247C16, KR200, KR331, KR625, KR707, KR959, KR1209, KR1369,KB2C-4, KB10H-5, KB456, KB42C17-13, KB45A43-3, KB54A33-1, KB58A10-3,KB59A54-4, KB59A54-5, KB53B7-8, KB53B7-2, KB60F5-7, KB60F5-11,KB59A58-4, KB60F5-15, KB61A18-1, KB65A15-2, KB65A15-3, KB65A15-7,KB65A15-8, KB65A15-12, KB65A14-1, KB3F-3, T25, KB53A71-6, KB65A11-2,KB68B57-1, KB63A5-3, and KB71A118-6.

Further identification and classification of novel toxin genes inpreparations of total genomic DNA was performed using the ³²P-labeledprobes and hybridization conditions described above in this Example.Total genomic DNA was prepared as above or with Qiagen Genomic-Tip 20/Gand Genomic DNA Buffer Set according to protocol for Gram positivebacteria (Qiagen Inc.; Valencia, Calif.) was used in southern analysis.For Southern blots, approximately 1-2 μg of total genomic DNA from eachstrain identified by slot blot analysis was digested with DraI and NdeIenzymes, electrophoresed on a 0.8% agarose gel, and immobilized on asupported nylon membrane using standard methods (Maniatis et al.). Afterhybridization, membranes were washed under low stringency (2×SSC/0.1%SDS at 42° C.) and exposed to film. DNA fragment sizes were estimatedusing BioRad Chemidoc system software. Restriction fragment lengthpolymorphisms were used to (arbitrarily) classify genes encoding the 44kDa toxin. These classifications are set forth in Table 12.

TABLE 12 RFLP Class (45 & 14 kD) Isolate Strain Name A 149B1  A′ KR331,KR1209, KR1369 B 167H2, 242K10 C 184M2, 201G, 201HH2 D 185GG, 187Y2,185FF1, 187F3 E 187G1 F 80JJ1, 186FF, 246P42 G 69Q H KB54A1-6 I KR136 JKR589 K 185L12, 185W3, 185Z11, 186L9, 187L14 L 147U2, 210B, KB10H-5,KB58A10-3, KB59A54-4, KB59A54-5, KB59A58-4, KB65A14-1 M 158T3, 158X10 N201H2, 201L3, 203G2, 203J1, 204C3, 204G4, 204I11, 204J7, 236B6 P 223L2,224F2  P′ 247C16, KB45A43-3, KB53B7-8, KB53B7-2, KB61A18-1, KB3F-3,KB53A71-6, KB6SA11- 2, KB68B57-1, KB63A5-3, KB71A118-6 Q 213E8,KB60F5-11, KB60F5-15 R KR959 S KB2C-4, KB46, KB42C17-13 T KB54A33-1,KB60F5-7 U T25 V KB65A15-2, KB65A15-3, KB65A15-7, KB65A15-8, KB65A15-12

EXAMPLE 17 DNA Sequencing of Additional Binary Toxin Genes

Degenerate oligonucleotides were designed to amplify all or part of the14 and 44.3 kDa genes from B.t. strains identified by hybridization withthe 149B1 PCR product described above. The oligonucleotides weredesigned to conserved sequence blocks identified by alignment of the 14kDa or 44.3 kDa genes from PS149B1, PS167H2 and PS80JJ1. Forward primersfor both genes were designed to begin at the ATG initiation codon.Reserve primers were designed as close to the 3′ end of each respectivegene as possible.

The primers designed to amplify the 14 kDa gene are as follows:

149DEG1 (forward): 5′-ATG TCA GCW CGY GAA GTW CAY ATT G-3′(SEQ ID NO:47) 149DEG2 (reverse): 5′-GTY TGA ATH GTA TAH GTH ACA TG-3′(SEQ ID NO:48)

These primers amplify a product of approximately 340 base pairs.

The primers designed to amplify the 44.3 kDa gene are as follows:

149DEG3 (forward): 5′-ATG TTA GAT ACW AAT AAA RTW TAT G -3′(SEQ ID NO:49) 149DEG4 (reverse): 5′-GTW ATT TCT TCW ACT TCT TCA TAH GAA G-3′ (SEQID NO: 50)

These primers amplify a product of approximately 1,100 base pairs.

The PCR conditions used to amplify gene products are as follows:

95° C., 1 min., one cycle

95° C., 1 min.

50° C., 2 min., this set repeated 35 cycles

72° C., 2 min.

72° C., 10 min., one cycle

PCR products were fractionated on 1% agarose gels and purified from thegel matrix using the Qiaexll kit (Qiagen). The resulting purifiedfragments were ligated into the pCR-TOPO cloning vector using the TOPOTA cloning kit (Invitrogen). After ligation, one half of the ligationreaction was transformed into XL10 Gold ultracompetant cells(Stratagene). Transformants were then screened by PCR with vectorprimers 1212 and 1233. Clones containing inserts were grown on theLB/carbenicillin medium for preparation of plasmids using the Qiagenplasmid DNA miniprep kit (Qiagen). Cloned PCR-derived fragments werethen sequenced using Applied Biosystems automated sequencing systems andassociated software. Sequences of additional novel binary toxin genesand polypeptides related to the holotype 14 and 44.3 kDa toxins fromPS80JJ1 and PS149B1 are listed as SEQ ID NOS. 51-126. The section above,entitled “Brief Description of the Sequences,” provides a furtherexplanation of these sequences.

The 14 kDa-type toxins and genes from three additional B.t. strains,PS137A, PS201V2 and PS207C3, were also sequenced using the aboveprocedures (with any differences noted below). PCR using the 149DEG1(forward) and 149DEG2 (reverse) primers was performed. These primersamplify a product of approximately 340 base pairs. The PCR was performedwith the following conditions:

1. 95° C., 3 min.

2. 94° C., 1 min.

3. 42° C., 2 min.

4. 72° C., 3 min.+5 sec./cycle

5. Steps 2 through 4 repeated 29 times

PCR products were gel purified using the QiaQuick gel extraction kit(Qiagen), the purified fragment was ligated into the pCR-TOPO cloningvector using the TOPO-TA kit (Invitrogen), and subsequently transformedinto XL10-Gold Ultracompetent E. coli cells (Strategene). Preparation oftransformant DNA is described above. Sequences of the 14 kDa toxin genefor each of the three new strains were obtained as per above. Thenucleotide and polypeptide sequences are provided in the attachedSequence Listing as follows: PS137A (SEQ ID NOs:149 and 150), PS201V2(SEQ ID NOs:151 and 152), and PS207C3 (SEQ ID NOs:153 and 154).

EXAMPLE 18 PS149B1 Toxin Transgenes and Plant Transformation

Separate synthetic transgenes optimized for maize codon usage weredesigned for both the 14 and 44.3 kDa toxin components. The syntheticversions were designed to modify the guanine and cytosine codon bias toa level more typical for plant DNA. Preferred plant-optimized transgenesare described in SEQ ID NOs:127-128. The promoter region used forexpression of both transgenes was the Zea mays ubiquitin promoter plusZ. mays exon 1 and Z. mays intron 1 (Christensen, A. H. et al. (1992)Plant Mol. Biol. 18:675-689). The transcriptional terminator used forboth transgenes was the potato proteinase inhibitor II (PinII)terminator (An, G. et al. 1989 Plant Cell 1:115-22).

Phosphinothricin acetyltransferase (PAT) was used as the selectablemarker for plant transformation. The phosphinothricin acetyltransferasegene (pat) was isolated from the bacterium Streptomycesviridochromogenes (Eckes P. et al., 1989). The PAT protein acetylatesphosphinothricin, or its precursor demethylphosphinothricin, conferringtolerance to a chemically synthesized phosphinothricin such as theherbicide glufosinate-ammonium. Acetylation converts phosphinothricin toan inactive form that is no longer toxic to corn plants. Glufosinateammonium is a broad spectrum, non-systemic, non-selective herbicide.Regenerating corn tissue or individual corn plants tolerant toglufosinate ammonium herbicide can be readily identified throughincorporation of PAT into regeneration medium or by spray application ofthe herbicide to leaves.

The synthetic version of the pat gene was produced in order to modifythe guanine and cytosine codon bias to a level more typical for plantDNA. The promoter for the pat gene is the CaMV promoter of the 35Stranscript from cauliflower mosiac virus (Pietrzak et al., 1986). Thetranscriptional terminator is the CaMV 35 S terminator.

For transformation of maize tissue, a linear portion of DNA, containingboth the PS149B1 14 and 44.3 kDa and pat selectable marker codingsequences, and the regulatory components necessary for expression, wasexcised from a complete plasmid. This linear portion of DNA, termed aninsert, was used in the transformation process.

Maize plants containing PS149B1 14 kDa and 44.3 kDa transgenes wereobtained by microprojectile bombardment using the Biolistics®Ò PDS-100Heparticle gun manufactured by Bio-Rad, essentially as described by Kleinet al. (1987). Immature embryos isolated from corn ears harvestedapproximately 15 days after pollination were cultured on callusinitiation medium for three to eight days. On the day of transformation,microscopic tungsten particles were coated with purified DNA andaccelerated into the cultured embryos, where the insert DNA wasincorporated into the cell chromosome. Six days after bombardment,bombarded embryos were transferred to callus initiation mediumcontaining glufosinate (Bialaphos) as the selection agent. Healthy,resistant callus tissue was obtained and repeatedly transferred to freshselection medium for approximately 12 weeks. Plants were regenerated andtransferred to the greenhouse. A total of 436 regenerated plants wereobtained. Leaf samples were taken for molecular analysis to verify thepresence of the transgenes by PCR and to confirm expression of theforeign protein by ELISA. Plants were then subjected to a whole plantbioassay using western corn rootworm. Positive plants were crossed withinbred lines to obtain seed from the initial transformed plants. Theseplants were found to be resistant to damage by corn rootworm in bothgreenhouse and field trials.

EXAMPLE 19 Further Bioassays

Protein preparations from the strains identified on Example 16 wereassayed for activity against western corn rootworm using the basic topload assay methods, as described in Example 13. The results are shown inTable 13.

TABLE 13 Strain LC₅₀ (ug/cm2) 95% Cl KB45A43-3 9.48 6.58-15.27 213′E′810.24 7.50-19.87 KR707 11.17  8.27-22.54# 185GG 11.53 7.51-16.81 187Y213.82 11.08-17.67  149B1 14.77 4.91-27.34 69Q 27.52 117.28-14.77#  167H231.38 19.35-47.60  KB54A33-10 32.62 24.76-83.85  185Z11 34.47 NDKB60F5-7 34.67 19.15-124.29 242K10 34.73 21.08-58.25  201G 34.90 13.20-355.18# 204J7 38.57 29.83-48.82  KB60F5-15 38.62   15.00-2.59E0380JJ1 41.96 27.35-139.43 203J1 43.85 23.18-69.51  KR589 47.28 29.83-230.71# 201HH2 49.94 23.83-351.77 KB60FS-11 51.84  19.38-1313.75#158X10 52.25 43.13-77.84# KB58A10-3 53.77 ND 201L3 55.01 41.01-78.96 158T3 58.07 39.59-211.13 184M2 60.54 26.57-411.88 204G4 69.0952.32-93.83  KB59A58-4 70.35 48.90-144.90 201H2 71.11 52.40-130.35 203G281.93 57.13-226.33 KB59A54-4 82.03   38.50-1.63E03 204I11 88.4162.48-173.07 236B6 89.33 64.16-158.96 KR1369 93.25  71.97-205.04#KB63A5-3 94.52 51.56-542.46 204C3 125.45  85.26-427.67# KR1209 128.1491.57-294.56 185W3 130.61 ND KR625 160.36 ND 210B 201.26     48.51-0.14E + 06# KB10H-5 214.25     87.97-8.22E + 03 KB68B57-1264.30      48.51-8.95E + 04# 223L2 3.81E + 02 ND KR136 7.83E + 02 — T251.30E + 03 ND KB61A18-1 2.58E + 03 ND 147U2 3.67E + 03 ND KR200 2.14E +05 ND KB59A54-5 3.32E + 05 ND KB3F-3 4.07E + 05 ND 187G1(bs) 3.50E + 07ND MR559 20%** n/a KB42C17-13 26%** n/a 224F2 33%** n/a KR959 41%** n/aKB2C-4 42%** n/a 198H3 46%** n/a KR331 47%** n/a KB46 55%** n/aKB71A118-6 71%** n/a KB53B7-2 84%** n/a 187Y2 ND n/a 185L12 ND ND 186L9ND n/a KB54A1-6 ND n/a 187L14 ND n/a 187G1(b) nt nt 187G1(s) nt nt

EXAMPLE 20 Molecular Cloning, Expression, and DNA Sequence Analysis of aNovel Binary Endotoxin Gene from Bacillus thuringiensis Strain PS201L3

Genomic DNA from PS201L3 was prepared from cells grown in shake flaskculture using a Qiagen Genomic-tip 500/G kit and Genomic DNA Buffer Setaccording to the protocol for Gram positive bacteria (Qiagen Inc.;Valencia, Calif.). A gene library was constructed from PS201L3 DNApartially digested with Sau3AI. Partial restriction digests werefractionated by agarose gel electrophoresis. DNA fragments 9.3 to 23 kbpin size were excised from the gel, electroeluted from the gel slice,purified on an Elutip-D ion exchange column (Schleicher and Schuell,Keene, N.H.), and recovered by ethanol precipitation. The Sau3AI insertswere ligated into BamHI-digested LambdaGem-11 (Promega, Madison, Wis.).Recombinant phage were packaged using Gigapack III XL Packaging Extract(Stratagene, La Jolla, Calif.) and plated on E. coli KW251 cells.Plaques were lifted onto Nytran Nylon Transfer Membranes (Schleicher &Schuell, Keene, N.H.) and probed with a ³²P-dCTP labeled gene probe forbinary toxin coding sequences. This gene probe was an approximately 1.0kb PCR product amplified using genomic PS201L3 DNA template andoligonucleotides “15kfor1” and “45krev6.”

15kfor1 (SEQ ID NO: 131) ATGTCAGCTCGCGAAGTACAC 45krev6 (SEQ ID NO: 132)GTCCATCCCATTAATTGAGGAG

The membranes were hybridized with the probe overnight at 65° C. andthen washed three times with 1×SSPE and 0.1% SDS. Thirteen plaques wereidentified by autoradiography. These plaques were subsequently pickedand soaked overnight in 1 mL SM Buffer+10 uL CHCl₃. Phage were platedfor confluent lysis on KW251 host cells; 6 confluent plates were soakedin SM and used for large-scale phage DNA preparations. The purifiedphage DNA was digested with various enzymes and run on 0.7% agarosegels. The gels were transferred to Nytran Membranes by Southern blottingand probed with the same PCR-amplified DNA fragment as above. Anapproximately 6.0 kb hybridizing XbaI band was identified and subclonedinto pHT370, an E. coli/Bacillus thuringiensis shuttle vector (Arantes,O., D. Lereclus [1991] Gene 108:115-119) to generate pMYC2476. XL10 GoldUltracompetent E. coli cells (Stratagene) transformed with pMYC2476 weredesignated MR1506. PMYC2476 was subsequently transformed intoacrytalliferous CryB cells by electroporation and selection onDM3+erythromycin (20 ug/mL) plates at 30° C. Recombinant CryB[pMYC2476]was designated MR561.

A subculture of MR1506 was deposited in the permanent collection of thePatent Culture Collection (NRRL), Regional Research Center, 1815 NorthUniversity Street, Peoria, Ill. 61604 USA on Jun. 1, 2000. The accessionnumber is B-30298.

B.t. strain MR561 was examined for expression of the PS201L3 binarytoxin proteins by immunoblotting. Cells were grown in liquid NYS-CAAmedium+erythromycin (10 ug/ml) overnight at 30° C. The culture was thenpelleted by centrifugation and a portion of the cell pellet wasresuspended and run on SDS-PAGE gels. Both 14 kDa and 44 kDa proteinswere apparent by Western Blot analysis when probed with antibodiesspecific for either the PS149B1 14 kDa or 44 kDa toxins, respectively.

DNA sequencing of the toxin genes encoded on pMYC2476 was performedusing an ABI377 automated sequencer. The DNA sequence for PS201L3 14 kDagene is shown in SEQ ID NO:133. The deduced peptide sequence for PS201L314 kDa toxin is shown in SEQ ID NO:134. The DNA sequence for PS201L3 44kDa gene is shown in SEQ ID NO:135. The deduced peptide sequence forPS201L3 44 kDa toxin is shown in SEQ ID NO:136.

The following table shows sequence similarity and identity of binarygenes and proteins from 201IL3 and 149B1. The program BESTFIT (part ofthe GCG software package) was used for these comparisons. BESTFIT usesthe local homology algorithm of Smith and Waterman (Advances in AppliedMathematics 2: 482-489 (1981)).

TABLE 14 201L3 vs 149B1 % similarity % identity 14 kDa nucleotide seq —71.1 14 kDa peptide seq 63.9 54.1 45 kDa nucleotide seq — 76.1 45 kDapeptide seq 70.9 62.7

EXAMPLE 21 Molecular Cloning and DNA Sequence Analysis of Novelδ-Endotoxin Genes From Bacillus thuringiensis Strains PS187G1, PS201HH2and KR1369

Total cellular DNA was prepared from Bacillus thuringensis strainsPS187G1, PS201HH2 and KR1369 grown to an optical density of 0.5-1.0 at600 nm visible light in Luria Bertani (LB) broth. DNA was extractedusing the Qiagen Genomic-tip 500/G kit and Genomic DNA Buffer Setaccording to the protocol for Gram positive bacteria (Qiagen Inc.;Valencia, Calif.). PS187G1, PS201HH2 and KR1369 cosmid libraries wereconstructed in the SuperCos1 vector (Stratragene) using inserts ofPS187G1, PS201HH2 and KR1369 total cellular DNA, respectively, partiallydigested with Nde II. XL1-Blue MR cells (Stratagene) were transfectedwith packaged cosmids to obtain clones resistant to carbenicillin andkanamycin. For each strain, 576 cosmid colonies were grown in 96-wellblocks in 1 ml LB+carbenicillin (100 ug/ml)+kanamycin (50 ug/ml) at 37°C. for 18 hours and replica plated onto nylon filters for screening byhybridization.

A PCR amplicon containing approximately 1000 bp of the PS187G1, PS201HH2or KR1369 14 kDa and 44 kDa toxin operon was amplified from PS187G1,PS201HH2 or KR1369 genomic DNA using primers designed to amplify binaryhomologs:

15kfor1: 5′-ATG TCA GCT CGC GAA GTA CAC-3′ (SEQ ID NO: 131) 45krev6:5′-GTC CAT CCC ATT AAT TGA GGA G-3′ (SEQ ID NO: 132)

The DNA fragment was gel purified using QiaQuick extraction (Qiagen).The probe was radiolabeled with ³²P-dCTP using the Prime-It II kit(Stratgene) and used in aqueous hybridization solution (6×SSPE,5×Denhardt's solution, 0.1% SDS, 0.1 mg/ml denatured DNA) with thecolony lift filters at 65° C. for 16 hours. The colony lift filters werebriefly washed 1× in 0.5×SSC/0.1%SDS at room temperature followed by twoadditional washes for 10 minutes at 65° C. in 0.5×SSC/0.1%SDS. Thefilters were then exposed to X-ray film for 20 minutes (PS187G1 andPS201HH2) or for 1 hour (KR1369). One cosmid clone that hybridizedstrongly to the probe was selected for further analysis for each strain.These cosmid clones were confirmed to contain the approximately 1000 bp14 kDa and 44 kDa toxin gene target by PCR amplification with theprimers listed above. The cosmid clone of PS187G1 was designated aspMYC3106; recombinant E. coli XL1-Blue MR cells containing pMYC3106 aredesignated MR1508. The cosmid clone of PS201HH2 was designated aspMYC3107; recombinant E. coli XL1-Blue MR cells containing pMYC3107 aredesignated MR1509. The cosmid clone of KR1369 was designated aspMYC3108; recombinant E. coli XL1-Blue MR cells containing pMYC3108 aredesignated MR1510. Subcultures of MR1509 and MR1510 were deposited inthe permanent collection of the Patent Culture Collection(NRRL),Regional Research Center, 1815 North University Street, Peoria,Ill. 61604 USA on Aug. 8, 2000. The accession numbers are NRRL B-30330and NRRL-B 30331, respectively.

The PS187G1, PS201HH2 and KR1369 14 kDa and 44 kDa toxin genes encodedby pMYC3106, pMYC3107 and pMYC3108, respectively, were sequenced usingthe ABI377 automated sequencing system and associated software.

The PS187G1 14 kDa and 44 kDa nucleotide and deduced polypeptidesequences are shown as SEQ ID NOs:137-140. Both the 14 kDa and 44 kDatoxin gene sequences are complete open reading frames. The PS187G1 14kDa toxin open reading frame nucleotide sequence, the 44 kDa toxin openreading frame nucleotide sequence, and the respective deduced amino acidsequences are novel compared to other toxin genes encoding pesticidalproteins.

The PS201HH2 14 kDa and 44 kDa nucleotide and deduced polypeptidesequences are shown as SEQ ID NOs:141-144. The 14 kDa toxin genesequence is the complete open reading frame. The 44 kDa toxin genesequence is a partial sequence of the gene. The PS201HH2 14 kDa toxinopen reading frame nucleotide sequence, the 44 kDa toxin partial openreading frame nucleotide sequence, and the respective deduced amino acidsequences are novel compared to other toxin genes encoding pesticidalproteins.

The KR1369 14 kDa and 44 kDa nucleotide and deduced polypeptidesequences are shown as SEQ ID NOs:145-148. Both the 14 kDa and 44 kDatoxin gene sequences are complete open reading frames. The KR1369 14 kDatoxin open reading frame nucleotide sequence, the 44 kDa toxin openreading frame nucleotide sequence, and the respective deduced amino acidsequences are novel compared to other toxin genes encoding pesticidalproteins.

EXAMPLE 22 Construction and Expression of a Hybrid Gene FusionContaining the PS149B1 14 kDa and 44 kDa Binary Toxin Genes

Oligonucleotide primers were designed to the 5′ and 3′ ends of both the14 kDa and 44 kDa genes from PS149B1. These oligonucleotides weredesigned to create a gene fusion by SOE-PCR (“Gene Splicing By OverlapExtension: Tailor-made Genes Using PCR,” Biotechniques 8:528-535, May1990). The two genes were fused together in the reverse order found inthe native binary toxin operon (i.e. 44 kDa gene first, followed by the14 kDa gene.)

The sequences of the olignucleotides used for SOE-PCR were thefollowing:

F1 new: AAATATTATTTTATGTCAGCACGTGAAGTACACATTG (SEQ ID NO: 155) R1 new:tctctGGTACCttaTTAtgatttatgcccatatcgtgagg (SEQ ID NO: 156) F2 new:agagaCTAGTaaaaaggagataaccATGttagatactaataaag (SEQ ID NO: 157) R2 new:CGTGCTGACATAAAATAATATTTTTAATTTTTTTAGTGTACTTT (SEQ ID NO: 158)

Oligo “F1new” was designed to direct amplification from the 5′ end ofthe 14 kDa gene and hybridize to the 3′ end of the 44 kDa gene. Oligo“R1new” was designed to direct amplification from the 3′ end of the 14kDa gene. This primer was designed with two stop codons in order toensure termination of translation. It was also designed with a KpnI sitefor directional cloning into a plasmid expression vector for Pseudomonasfluorescens. Oligo “F2new” was designed to direct amplification from the5′ end of the 44 kDa gene. It also includes a ribosome binding sequenceand a SpeI cloning site. Oligo “R2new” was designed to directamplification from the 3′ end of the 44 kDa gene and hybridize to the 5′end of the 14 kDa gene.

The two genes were first independently amplified from PS149B1 genomicDNA; the 14 kDa gene using “F1new” and “R1new,” and the 44 kDa geneusing “F2new” and “R2new.” The products were then combined in one PCRtube and amplified together using “R1new” and “F2new.” At this point,Herculase™ Enhanced Polymerase Blend (Stratagene, La Jolla, Calif.) wasused at a 48° C. annealing temperature to amplify a 1.5 kb DNA fragmentcontaining the gene fusion. This DNA fragment was subsequently digestedusing KpnI and SpeI, fractionated on agarose gels, and purified byelectroelution. The plasmid vector was also digested with KpnI and SpeI,fractionated on agarose gels, purified by electroelution and treatedwith phosphatase. The vector and insert were then ligated togetherovernight at 14° C. Ligated DNA fragments were transformed into MB214P.f. cells by electroporation and selection overnight on LB+tetracycline (30 ug/mL) plates. Strains containing the gene fusion wereidentified by diagnostic PCR and sequenced for verification ofsuccessful gene splicing. One representative strain containing thecloned gene fusion was designated MR1607; the recombinant plasmid wasdesignated pMYC2475.

A subculture of MR1607 was deposited in the permanent collection of thePatent Culture Collection (NRRL), Regional Research Center, 1815 NorthUniversity Street, Peoria, Ill. 61604 USA on Aug. 8, 2000. The accessionnumber is NRRL B-30332. MR1607 was grown and protein production wasverified by SDS-PAGE and immunoblotting. A protein band at ˜58 kDarepresenting the 44 kDa+14 kDa fusion product was identified whenwestern blots were probed with antibodies specific to either the 14 kDatoxin or the 44 kDa toxin.

The sequence of the 58 kDa fusion protein is provided in SEQ ID NO:159.The DNA sequence for the gene fusion is provided in SEQ ID NO:160.

EXAMPLE 23 Binary Homologue Mixing Study

Growth of Homologue Strains.

Four strains were selected, one from each major binary toxinfamily—149B1 , 80JJ1, 201L3, and 167H2. In order to reduce time spentpurifying individual toxin proteins, the following Pseudomonasfluorescens (P.f.) clones were grown instead: MR1253 (14 kDa of 149B1)and MR1256 (44 kDa of 149B1). Similarly, B.t. clones MR541 (expressing14 kDa of 80JJ1), and MR542 (44 kDa of 80JJ1) were used. B.t. strainswere grown as described in Example 1. Pellets were washed 3× with waterand stored at −20° C. until needed. P.f. strains were grown in 10 Lbatches in Biolafitte fermenters using standard procedures. Pellets werestored at −80° C. until needed.

Extraction & Purification of Toxins.

Purification of 167H2, MR541, MR542, 201L3. Extractions of cell pelletswere done using 100 mM sodium citrate buffer at pH's ranging from 3.0 to5.5. In a typical extraction, pellets were extracted with a buffervolume {fraction (1/10)} to ⅓× of the original culture volume. Pelletswere suspended in the buffer and placed on a rocking platform at 4° C.for periods of time ranging from 2.5 hours to overnight. The extractswere centrifuged and supernatants were retained. This procedure wasrepeated with each strain until at least approximately 10 mg of eachprotein were obtained. SDS-PAGE confirmed the presence/absence ofprotein toxins in the extracts through use of the NuPAGE Bis/Tris gelsystem (Invitrogen). Samples were prepared according to themanufacturers instructions and were loaded onto 4-12% gels and theelectrophoretograms were developed with MES running buffer. Theexception to this procedure was the sample prep of all 201L3 samples.These samples were prepared by diluting ½× with BioRad's Laemmli samplebuffer and heating at 95° C. for 4 minutes. Protein quantitation wasdone by laser scanning gel densitometry with BSA as a standard(Molecular Dynamics Personal Densitometer SI). Extracts were clarifiedby filtration through a 0.2 μm membrane filter and stored at 4° C.

Purification of MR1253 & MR1256. The recombinant proteins MR1253 and MR1256, corresponding to the 14 and 44 kDa proteins of 149B1 respectively,were prepared as solubilized inclusions. Inclusion bodies were preparedusing standard procedures. The inclusion bodies were solubilized in 1 mMEDTA, 50 mM sodium citrate, pH 3.5.

Purification of individual toxins, 167H2 & 201L3. All extracts known tocontain either the 14, the 44 kDa, or both were combined. This combinedextract was dialyzed against 100 mM sodium citrate, 150 mM NaCl, pH 4.Dialysis tubing was from Pierce (Snakeskin 10 k MWCO). Samples wereusually dialyzed for approximately 6 hours and then again overnight infresh buffer.

Extracts were then concentrated with either Centriprep 10 or CentriconPlus-20 (Biomax—5, 5000 NMWL) centrifugal filter devices (Millipore),quantitated for both the 14 kDa and 44 kDa proteins, and subjected togel filtration chromatography.

In preparation for chromatography, all samples and buffers were filteredthrough a 0.2 μm filter and degassed. Samples were then applied to aHiPrep 26/60 Sephacryl S-100 gel filtration column which had beenequilibrated with two bed volumes of the separation buffer, 100 mMsodium citrate, 150 mM NaCl, pH 4.0. Sample volumes ranged from 5-10 ml.An AKTA purifier 100 FPLC system (Amersham Pharmacia) controlled theruns. Chromatography was done at ambient temperature. Buffer flowthrough the column during the run was maintained at 0.7 ml/min. Proteinswere detected by monitoring UV absorbance at 280 nm. Fractions werecollected and stored at 4° C. Fractions containing either the 14 or 44kDa protein were pooled and checked for purity by SDS-PAGE as describedabove.

For 167H2 samples, two large peaks were detected and were well separatedfrom each other at the baseline. SDS-PAGE of fractions showed each peakrepresented one of the protein toxins.

In the 201L3 sample, three well defined peaks and one shoulder peak weredetected. SDS-PAGE revealed that the first peak represented a 100 kDaprotein plus an 80 kDa protein. The second peak represented the 44 kDaprotein, while the shoulder peak was a 40 kDa protein. The third peakwas the 14 kDa protein. Fractions with the 44 kDa from both samples werecombined as were all fractions containing the 14 kDa.

The 149B1 proteins had been obtained individually from Pf clones MR1253and MR1256 and, therefore, further purification was not necessary.Similarly, the 80JJ1 recombinants, MR541 and MR542 yielded theindividual 14 and 44 kDa proteins thereby obviating furtherpurification.

Sample Preparation for wCRW LC₅₀ Bioassay.

Dialysis. Samples of individual binary toxin proteins were dialyzedagainst 6 L of 20 mM sodium citrate, pH 4.0. The first dialysisproceeded for several hours, the samples were transferred to freshbuffer and alowed to dialyze overnight. Finally, the samples weretransferred to fresh buffer and dialyzed several more hours. Sources ofthe protein samples were either the pooled gel-filtration fractions(167H2, 201L3), pellet extracts (MR541, MR542), or inclusion pelletextracts (MR1253, MR1256). All samples were filtered through 0.2 ummembranes to sterilize.

Concentration. Samples were concentrated with Centricon Plus-20(Biomax—5, 5000 NMWL) centrifugal filter devices (Millipore).

Quantitation. Samples were quantitated for protein as above. To meet therequirements of the LC₅₀ bioassay, a minimum of 6.3 mg of each toxinprotein were needed at a concentration range of 0.316-1.36 mg/ml for thevarious combinations. If necessary, samples were concentrated as above,or were diluted with buffer (20 mM sodium citrate, pH 4.0) andrequantitated.

Mixing of binaries/LC₅₀ bioassay. For each of the four strains, the 14kDa was combined with an amount the 44 kDa of each strain to give a 1/1mass ratio. The top dose was 50 ug/cm² for the mixtures, with theexception of mixtures with the 14 kDa protein of 203J1. Top doses ofmixtures with this protein were only 44 ug/cm². For controls, eachprotein was submitted individually, as was the extract buffer, 20 mMsodium citrate, pH 4.0. Native combinations were also tested (i.e. 14kDa+44 kDa of 149B1). All toxin combinations and buffer controls wereevaluated three times by bioassay against Western corn rootworm, whileindividual toxins were tested once.

The results are reported below in Table 15 (LC₅₀ Results for ToxinCombinations) and Table 16 (Comparison of Potencies of Strains to149B1).

TABLE 15 Toxin combination Top load, ul/well LC₅₀ (ug/cm²) 80JJ1 14 +80JJ1 44 96 28 (19-44 C.I.) 167H2 44 159 >Top dose 201L3 44 172 No doseresponse 149B1 44 78 No dose response 167H2 14 + 167H2 44 161 19 (13-27C.I.) 80JJ1 44 97 No dose response 201L3 44 174 14 (10-22 C.I.) 149B1 4480 No dose response 201L3 14 + 201L3 44 193 No dose response 80JJ1 44116 No dose response 167H2 44 180 No dose response 149B1 44 99 No doseresponse 149B1 14 + 149B1 44 45 10 (7-15 C.I.) 80JJ1 44 63 11 (8-16C.I.) 167H2 44 126 8 (6-11 C.I.) 201L3 44 139 18 (13-27 C.I.)

TABLE 16 Comparison of potencies of strains to 149B1 Toxin combinationRelative potency 149B1 14 + 149B1 44 To which all others are compared149B1 14 + 80JJ1 44 0.9 149B1 14 + 167H2 44 1.3 149B1 14 + 201L3 44 0.580JJ1 14 + 80JJ1 44 0.4 167H2 14 + 167H2 44 0.5 167H2 14 + 201L3 44 0.7

The results are also displayed graphically in FIG. 3.

Native combinations were highly active against Western corn rootworm,except for 201L3. However, the 44 kDa of 201L3 was active when combinedwith either the 14 kDa of 167H2 or 149B1. Other active combinations werethe 149B1 14 kDa with either 80JJ1 or 167H2 44 kDa, with the latterappearing to be more active than the native 149B1 mixture. No doseresponse was noted for either the individual proteins, or the buffer andwater controls.

EXAMPLE 24 Control of Southern Corn Rootworm With PS149B1 14-kDa Protein

A powder containing approximately 50% (wt/wt) of a 14-kDa δ-endotoxin,originally discovered in Bacillus thuringiensis strain PS149B1, wasisolated from recombinant Pseudomonas fluorescens strain (MR1253). Thispowder was evaluated for insecticidal activity using the followingprocedure.

Artificial insect diet (R. I. Rose and J. M. McCabe (1973), “Laboratoryrearing techniques for rearing corn rootworm,” J. Econ. Entomol. 66(2):398-400) was dispensed at ˜0.5 mL/well into 128-well bioassay trays (C-DInternational, Pitman, N.J.) to produce a surface area of ˜1.5 cm2.Buffer (10-mM potassium phosphate, pH 7.5) suspensions of the 14-kDaprotein powder were applied to the surface of the artificial insect dietat 50 μL/well, and the diet surface was allowed to dry. Buffer controlswere also included in each test. A single neonate southern cornrootworn, Diabrotica undecimpunctata howardi, was placed in each well,and the wells were sealed with lids that were provided with the trays.The bioassays were held for 6 days at 28° C., after which time, the livelarvae were weighed as a group for each treatment. Percent growthinhibition was calculated by subtracting the weight of live insects fromeach treatment from the weight of live, control insects, and thendividing by the control weight. This result was multiplied by 100 toconvert the number to a percent. Growth inhibition was calculated foreach of 5 tests that each contained 16 insects per treatment, and thegrowth inhibition was averaged across tests.

Results demonstrated that the 14-kDa protein inhibited growth ofsouthern corn rootworms in a concentration-dependent manner. Table 17shows southern corn rootworn growth inhibition with PS149B1 14-kDaprotein.

TABLE 17 Treatment Concentration in μg ai/cm² % Growth Inhibition 14-kDaProtein 1 32 14-kDa Protein 3 55 14-kDa Protein 9 78 ai = activeingredient

EXAMPLE 25 Control of European Corn Borer and Corn Earworm With PS149B1Binary Toxin

A powder containing 54% of a 14-kDa δ-endotoxin, and another powdercontaining 37% of a 44-kDa δ-endotoxin, both originally discovered inBacillus thuringiensis strain PS149B1, were isolated from recombinantPseudomonas fluorescens strains MR1253 and MR1256, respectively.Mixtures of these powders were evaluated for insecticidal activity usingthe following procedure.

Artificial insect diet (R. I. Rose and J. M. McCabe (1973), “Laboratoryrearing techniques for rearing corn rootworm,” J. Econ. Entomol. 66(2):398-400) was dispensed at ˜0.5 mL/well into 128-well bioassay trays (C-DInternational, Pitman, N.J.) to, produce a surface area of ˜1.5 cm2.Buffer (10-mM potassium phosphate, pH 7.5) suspensions of the proteinpowders were mixed, and were then applied to the surface of theartificial insect diet at 50 μL/well. The diet surface was allowed todry. Buffer controls were also included in each test. A single neonatelarvae was placed in each well, and the wells were sealed with lids thatwere provided with the trays. Tests were conducted with European cornborer, Ostrinia nubilalis, and corn earworm, Helicoverpa zea (both arelepidopterans). The bioassays were held for 6 days at 28° C., afterwhich time, the live larvae were weighed as a group for each treatment.Percent growth inhibition was calculated by subtracting the weight oflive insects in each treatment from the weight of live, control insects,and then dividing by the control weight. This result was multiplied by100 to convert the number to a percent. Growth inhibition was calculatedfor each of 4 tests that each contained 14 to 16 insects per treatment,and the growth inhibition was averaged across tests.

Results demonstrated that the 14-kDa protein inhibited growth ofEuropean corn borers and corn earworms in a concentration-dependentmanner. Table 18 shows corn earworm (CEW) and European corn borer (ECB)growth inhibition with PS149B1 protein mixtures.

TABLE 18 14-kDa protein + 44-kDa Protein % Growth InhibitionConcentration in μg ai/cm² CEW ECB 3.7 + 11  42 59 11 + 33 57 77  33 +100 61 89 ai = active ingredient

EXAMPLE 26 Further Characterization of the 45 kDa Proteins and PrimerDesign for Identifying Additional Polynucleotides and Proteins

The subject invention includes not only the specifically exemplifiedsequences. Portions of the subject genes and toxins can be used toidentify other related genes and toxins. Thus, the subject inventionincludes polynucleotides that encode proteins or polypeptides comprisingat least ten contiguous amino acids, for example, of any of thebinary-type proteins or polypeptides included in the attached sequencelisting and described herein. Other embodiments include polynucleotidesthat encode, for example, at least 20, 30, 40, 50, 60, 70, 80, 90, and100 contiguous amino acids of a protein exemplified herein; thesenumbers also apply similarly to contiguous nucleotides of an exemplifiedpolynucleotide. The proteins encoded by such polynucleotides areincluded in the subject invention. Likewise, polynucleotides comprisingcontiguous nucleotides (that code for proteins or polypeptidescomprising peptides of these approximate sizes) are included in thesubject invention.

While still very different, the “closest” toxins to those of the subjectinvention are believed to be the 51 and 42 kDa mosquitocidal proteins ofBacillus sphaericus. Attached as FIGS. 4 and 5 are protein alignmentsand nucleotide sequences alignments of the 51 and 42 kDa sphaericustoxins and genes and the 45 kDa 149B1 toxin and gene.

Two blocks of sequences are highlighted in the nucleotide alignment towhich primers could be made. An exemplary PCR primer pair is includedbelow, and in 5′-3′ orientation (45 kD3′rc is shown as the complement).These primers have been successfully used to identify additional membersof the 45 kDa binary family. Fully redundant sequences and a propheticpair are also included below.

45kD5′: GAT RAT RAT CAA TAT ATT ATT AC (SEQ ID NO: 161). 45kD3′rc: CAAGGT ART AAT GTC CAT CC (SEQ ID NO: 162).

The sequences would be useful as both the sequence written and as thereverse complement (03 and 04 are complementary to 45 kD3′rc, theexemplified reverse primer).

45kD5′01: GAT GATGrTmrAk wwATTATTrC A (SEQ ID NO: 163). 45kD5′02: GATGATGrTmrAT ATATTATTrC A (SEQ ID NO: 164). 45kD3′03: GGAwG krCdyTwdTmCCwTGTAT (SEQ ID NO: 165). 45kD3′04: GGAwG kACryTAdTA CCTTGTAT (SEQ IDNO: 166).

Regarding the manner in which the sphaericus toxins were identified, aBLAST (Altschul et al. (1997), “Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programs,” Nucleic Acids Res.25:3389-3402) database search using the 149B1 45 kDa protein foundmatches to the 42 kDa B. sphaericus crystal inclusion protein(expectation score 3*10⁻⁴) and the 51 kDa B. sphaericus crystalinclusion protein (expectation score 3*10⁻⁹).

An alignment of the 45 kDa 149B1 peptide sequence to the 42 kDa B.sphaericus crystal inclusion protein results in an alignment having 26%identity over 325 residues. The alignment score is 27.2 sd above themean score of 100 randomized alignments. A similar analysis of the 45kDa 149B1 peptide sequence to the 42 kDa B. sphaericus crystal inclusionprotein results in an alignment having 29% identity over 229 residues.The alignment score is 23.4 sd above the mean score of 100 randomizedalignments. Alignment scores>10 sd above the mean of random alignmentshave been considered significant (Lipman, D. J. and Pearson, W. R.(1985), “Rapid and sensitive similarity searches,” Science227:1435-1441; Doolittle, R. F. (1987), Of URFs and ORFs: a primer onhow to analyze derived amino acid sequences, University Science Books,Mill Valley, Calif.).

For reference, the structurally similar Cry1Aa, Cry2Aa and Cry3Aaprotein sequences were compared in the same way. Cry2Aa vs. Cry1Aa andCry2Aa vs. Cry3Aa share 29% and 27% identity over 214 and 213 residues,respectively, with alignment scores 32.2 sd and 29.5 sd above the meanscore of 100 randomized alignments. An alignment of the 149B1 45 kDaprotein sequence and the Cry2Aa protein sequence resulted in analignment score within 1 sd of the mean of 100 randomized alignments.

The following comparisons are also noted:

TABLE 19 % % Average Comparison Quality Length Ratio Gaps SimilarityIdentity Quality* ps149b1-45.pep × 189 325 0.612 12 35.135 26.351 39.4 ±5.5 s07712 ps149b1-45.pep × 161 229 0.742 9 36.019 28.910 39.3 ± 5.207711 cry2aa1.pep × 182 214 0.888 6 37.688 28.643 43.5 ± 4.3 cry1aa1.pepcry3aa1.pep × 187 213 0.926 6 40.500 27.000 42.3 ± 4.9 cry2aa1.pepps149b1-45.pep × 40 28 1.429 0 42.857 35.714 41.6 ± 5.6 cry2aa1.pep*based on 100 randomizations

For further comparison purposes, and for further primer design, thefollowing references are noted:

Oei et al. (1992), “Binding of purified Bacillus sphaericus binary toxinand its deletion derivatives to Culex quinquefasciatus gut: elucidationof functional binding domains,” Journal of General Microbiology 138(7):1515-26.

For the 51 kDa: 35-448 is active; 45-448 is not; 4-396 is active; 4-392is not.

For the 42 kDa: 18-370 is active, 35-370 is not; 4-358 is active; 4-349is not.

The work was done with GST fusions purified and cleaved with thrombin.All truncations were assayed with==of other intact subunit. Alldeletions had some loss of activity. P51deltaC56 binds, but doesn'tinternalize 42. P51delta N45 doesn't bind. Only 42 kDa+51 kDa areinternalized. Both N-terminal and C-terminal non-toxic 42 kDa proteinsfailed to bind the 51 kDa protein or 51 kDa-receptor complex.

Davidson et al. (1990), “Interaction of the Bacillus sphaericus mosquitolarvicidal proteins,” Can. J. Microbiol. 36(12):870-8. N-termini ofSDS-PAGE purified proteins obtained from B. sphaericus. S29 and N31 of51 kDa and S9 of 42 kDa in 68-74 kDa complexes (unreduced). S9 and S29of 51 and N31 of 42 from 51 kDa band (unreduced). In reduced gels the 45kDa band had S29 and N31 of the 51 kDa and the 39 kDa band contained S9of the 42 kDa protein.

Baumann et al. (1988), “Sequence analysis of the mosquitocidal toxingenes encoding 51.4- and 41.9-kilodalton proteins from Bacillussphaericus 2362 and 2297,” J. Bacteriol. 17:2045-2050. N-termini of 41.9kDa at D5 from B. sphaericus protease and I11 from chymotrypsin;C-terminus following R349 with trypsin. Regions of enhanced similaritywere identified that correspond to many of those above. Similar sequenceblocks A through D between the 51 and 42 kDa proteins.

In summary, the sphaericus toxins discussed above are not meant to beincluded in the scope of the subject invention (in fact, they arespecifically excluded). In that regard, divergent contiguous sequences,as exemplified in the alignments (FIGS. 4 and 5) discussed above, can beused as primers to identify unique toxins that are suggested but notspecifically exemplified herein. However, the conserved contiguoussequences, as shown in the alignments, can also be used according to thesubject invention to identify further novel 15/45 kDa-type binary toxins(active against corn rootworm and other pests).

EXAMPLE 27 Insertion and Expression of Toxin Genes in Plants

One aspect of the subject invention is the transformation of plants withpolynucleotides of the subject invention that express proteins of thesubject invention. The transformed plants are resistant to attack by thetarget pest.

The novel corn rootworm-active genes described here can be optimized forexpression in other organisms. For example, maize optimized genesequences encoding the 14 and 44 kDa PS80JJ1 toxins are disclosed in SEQID NO:44 and SEQ ID NO:45, respectively.

Genes encoding pesticidal toxins, as disclosed herein, can be insertedinto plant cells using a variety of techniques which are well known inthe art. For example, a large number of cloning vectors comprising areplication system in E. coli and a marker that permits selection of thetransformed cells are available for preparation for the insertion offoreign genes into higher plants. The vectors comprise, for example,pBR322, pUC series, M13mp series, pACYC184, etc. Accordingly, thesequence encoding the B.t. toxin can be inserted into the vector at asuitable restriction site. The resulting plasmid is used fortransformation into E. coli. The E. coli cells are cultivated in asuitable nutrient medium, then harvested and lysed. The plasmid isrecovered. Sequence analysis, restriction analysis, electrophoresis, andother biochemical-molecular biological methods are generally carried outas methods of analysis. After each manipulation, the DNA sequence usedcan be cleaved and joined to the next DNA sequence. Each plasmidsequence can be cloned in the same or other plasmids. Depending on themethod of inserting desired genes into the plant, other DNA sequencesmay be necessary. If, for example, the Ti or Ri plasmid is used for thetransformation of the plant cell, then at least the right border, butoften the right and the left border of the Ti or Ri plasmid T-DNA, hasto be joined as the flanking region of the genes to be inserted.

The use of T-DNA for the transformation of plant cells has beenintensively researched and sufficiently described in EP 120 516; Hoekema(1985) In: The Binary Plant Vector System, Offset-durkkerij Kanters B.V., Alblasserdam, Chapter 5; Fraley et al., Crit. Rev. Plant Sci.4:1-46; and An et al. (1985) EMBO J. 4:277-287.

Once the inserted DNA has been integrated in the genome, it isrelatively stable there and, as a rule, does not come out again. Itnormally contains a selection marker that confers on the transformedplant cells resistance to a biocide or an antibiotic, such as kanamycin,G 418, bleomycin, hygromycin, or chloramphenicol, inter alia. Theindividually employed marker should accordingly permit the selection oftransformed cells rather than cells that do not contain the insertedDNA.

A large number of techniques are available for inserting DNA into aplant host cell. Those techniques include transformation with T-DNAusing Agrobacterium tumefaciens or Agrobacterium rhizogenes astransformation agent, fusion, injection, biolistics (microparticlebombardment), or electroporation as well as other possible methods. IfAgrobacteria are used for the transformation, the DNA to be inserted hasto be cloned into special plasmids, namely either into an intermediatevector or into a binary vector. The intermediate vectors can beintegrated into the Ti or Ri plasmid by homologous recombination owingto sequences that are homologous to sequences in the T-DNA. The Ti or Riplasmid also comprises the vir region necessary for the transfer of theT-DNA. Intermediate vectors cannot replicate themselves in Agrobacteria.The intermediate vector can be transferred into Agrobacteriumtumefaciens by means of a helper plasmid (conjugation). Binary vectorscan replicate themselves both in E. coli and in Agrobacteria. Theycomprise a selection marker gene and a linker or polylinker which areframed by the right and left T-DNA border regions. They can betransformed directly into Agrobacteria (Holsters et al. [1978] Mol. Gen.Genet. 163:181-187). The Agrobacterium used as host cell is to comprisea plasmid carrying a vir region. The vir region is necessary for thetransfer of the T-DNA into the plant cell. Additional T-DNA may becontained. The bacterium so transformed is used for the transformationof plant cells. Plant explants can advantageously be cultivated withAgrobacterium tumefaciens or Agrobacterium rhizogenes for the transferof the DNA into the plant cell. Whole plants can then be regeneratedfrom the infected plant material (for example, pieces of leaf, segmentsof stalk, roots, but also protoplasts or suspension-cultivated cells) ina suitable medium, which may contain antibiotics or biocides forselection. The plants so obtained can then be tested for the presence ofthe inserted DNA. No special demands are made of the plasmids in thecase of injection and electroporation. It is possible to use ordinaryplasmids, such as, for example, pUC derivatives.

The transformed cells grow inside the plants in the usual manner. Theycan form germ cells and transmit the transformed trait(s) to progenyplants. Such plants can be grown in the normal manner and crossed withplants that have the same transformed hereditary factors or otherhereditary factors. The resulting hybrid individuals have thecorresponding phenotypic properties.

In a preferred embodiment of the subject invention, plants will betransformed with genes wherein the codon usage has been optimized forplants. See, for example, U.S. Pat. No. 5,380,831, which is herebyincorporated by reference. Also, advantageously, plants encoding atruncated toxin will be used. The truncated toxin typically will encodeabout 55% to about 80% of the full length toxin. Methods for creatingsynthetic B.t. genes for use in plants are known in the art.

EXAMPLE 28 Cloning of B.t. Genes Into Insect Viruses

A number of viruses are known to infect insects. These viruses include,for example, baculoviruses and entomopoxviruses. In one embodiment ofthe subject invention, genes encoding the insecticidal toxins, asdescribed herein, can be placed within the genome of the insect virus,thus enhancing the pathogenicity of the virus. Methods for constructinginsect viruses which comprise B.t. toxin genes are well known andreadily practiced by those skilled in the art. These procedures aredescribed, for example, in Merryweather et al. (Merryweather, A. T., U.Weyer, M. P. G. Harris, M. Hirst, T. Booth, R. D. Possee (1990) J. Gen.Virol. 71:1535-1544) and Martens et al. (Martens, J. W. M., G. Honee, D.Zuidema, J. W. M. van Lent, B. Visser, J. M. Vlak (1990) Appl.Environmental Microbiol. 56(9):2764-2770).

All patents, patent applications, provisional applications, andpublications referred to or cited herein are incorporated by referencein their entirety to the extent they are not inconsistent with theexplicit teachings of this specification.

It should be understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to be included within the spirit and purview of thisapplication and the scope of the appended claims.

166 1 5 PRT Bacillus thuringiensis 1 Met Leu Asp Thr Asn 1 5 2 25 PRTBacillus thuringiensis 2 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser AsnLeu Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu 20 25 3 24PRT Bacillus thuringiensis 3 Ser Ala Arg Glu Val His Ile Glu Ile Asn AsnThr Arg His Thr Leu 1 5 10 15 Gln Leu Glu Ala Lys Thr Lys Leu 20 4 25PRT Bacillus thuringiensis 4 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile SerAsn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu 20 255 50 PRT Bacillus thuringiensis MISC_FEATURE (35) Undetermined aminoacid 5 Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 15 10 15 Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg Thr20 25 30 Ser Pro Xaa Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala Glu35 40 45 Ser Asn 50 6 25 PRT Bacillus thuringiensis 6 Met Leu Asp ThrAsn Lys Ile Tyr Glu Ile Ser Asn Tyr Ala Asn Gly 1 5 10 15 Leu His AlaAla Thr Tyr Leu Ser Leu 20 25 7 25 PRT Bacillus thuringiensis 7 Ser AlaArg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 1 5 10 15 LeuGln Leu Glu Asp Lys Thr Lys Leu 20 25 8 29 DNA Artificial SequenceDescription of Artificial Sequence oligonucleotide probe for geneencoding PS80JJ1 44.3 kDa toxin; forward primer for PS149B1 and PS167H28 atgntngata cnaataaagt ntatgaaat 29 9 26 DNA Artificial SequenceDescription of Artificial Sequence reverse primer for PS149B1 andPS167H2 9 ggattatcta tctctgagtg ttcttg 26 10 1158 DNA Bacillusthuringiensis 10 atgttagata ctaataaagt ttatgaaata agcaatcttg ctaatggattatatacatca 60 acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaaggatgaagatatt 120 gatgattaca atttaaaatg gtttttattt cctattgata ataatcaatatattattaca 180 agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaataaatgtttca 240 acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaagattcttcatat 300 ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatctcttggaata 360 gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaatttaactcctgta 420 caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaagatcatcctgaa 480 tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatgggatggacatta 540 gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaattaaaactact 600 ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaagtaatgtatct 660 ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaaaaatcaaaaa 720 acaactatta ttaatacagt aggattgcaa attaatatag attcaggaatgaaatttgaa 780 gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactgaagaattaaaa 840 gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactcagagatagat 900 aatccaacta atcaaccaat gaattctata ggacttctta tttatacttctttagaatta 960 tatcgatata acggtacaga aattaagata atggacatag aaacttcagatcatgatact 1020 tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcacaaaccattcg 1080 tatgaagaag tagaagaaat aacaaaaata cctaagcata cacttataaaattgaaaaaa 1140 cattatttta aaaaataa 1158 11 385 PRT Bacillusthuringiensis 11 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu AlaAsn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser GlyVal Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn LeuLys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr SerTyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys Asn Asp Lys IleAsn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp GlnIle Lys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly LysVal Leu Thr Ala 100 105 110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg LeuThr Asp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu ThrPro Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp GluLys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser Glu Thr Gly Asn IleAsn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val ProCys Ile Met Val Asn Asp Ser Lys Ile Asp 180 185 190 Lys Asn Thr Gln IleLys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp AsnLeu Ala Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220 Gln Lys ArgSer Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230 235 240 ThrThr Ile Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275280 285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn290 295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu GluLeu 305 310 315 320 Tyr Arg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp IleGlu Thr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro AsnHis Lys Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu GluVal Glu Glu Ile Thr 355 360 365 Lys Ile Pro Lys His Thr Leu Ile Lys LeuLys Lys His Tyr Phe Lys 370 375 380 Lys 385 12 834 DNA Bacillusthuringiensis 12 ggactatatg cagcaactta tttaagttta gatgattcag gtgttagtttaatgaataaa 60 aatgatgatg atattgatga ttataactta aaatggtttt tatttcctattgatgatgat 120 caatatatta ttacaagcta tgcagcaaat aattgtaaag tttggaatgttaataatgat 180 aaaataaatg tttcgactta ttcttcaaca aattcaatac aaaaatggcaaataaaagct 240 aatggttctt catatgtaat acaaagtgat aatggaaaag tcttaacagcaggaaccggt 300 caagctcttg gattgatacg tttaactgat gaatcctcaa ataatcccaatcaacaatgg 360 aatttaactt ctgtacaaac aattcaactt ccacaaaaac ctataatagatacaaaatta 420 aaagattatc ccaaatattc accaactgga aatatagata atggaacatctcctcaatta 480 atgggatgga cattagtacc ttgtattatg gtaaatgatc caaatatagataaaaatact 540 caaattaaaa ctactccata ttatatttta aaaaaatatc aatattggcaacgagcagta 600 ggaagtaatg tagctttacg tccacatgaa aaaaaatcat atacttatgaatggggcaca 660 gaaatagatc aaaaaacaac aattataaat acattaggat ttcaaatcaatatagattca 720 ggaatgaaat ttgatatacc agaagtaggt ggaggtacag atgaaataaaaacacaacta 780 aatgaagaat taaaaataga atatagtcat gaaactaaaa taatggaaaaatat 834 13 278 PRT Bacillus thuringiensis 13 Gly Leu Tyr Ala Ala ThrTyr Leu Ser Leu Asp Asp Ser Gly Val Ser 1 5 10 15 Leu Met Asn Lys AsnAsp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp 20 25 30 Phe Leu Phe Pro IleAsp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala 35 40 45 Ala Asn Asn Cys LysVal Trp Asn Val Asn Asn Asp Lys Ile Asn Val 50 55 60 Ser Thr Tyr Ser SerThr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala 65 70 75 80 Asn Gly Ser SerTyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr 85 90 95 Ala Gly Thr GlyGln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser 100 105 110 Ser Asn AsnPro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile 115 120 125 Gln LeuPro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro 130 135 140 LysTyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu 145 150 155160 Met Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile 165170 175 Asp Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys180 185 190 Tyr Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu ArgPro 195 200 205 His Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu IleAsp Gln 210 215 220 Lys Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile AsnIle Asp Ser 225 230 235 240 Gly Met Lys Phe Asp Ile Pro Glu Val Gly GlyGly Thr Asp Glu Ile 245 250 255 Lys Thr Gln Leu Asn Glu Glu Leu Lys IleGlu Tyr Ser His Glu Thr 260 265 270 Lys Ile Met Glu Lys Tyr 275 14 829DNA Bacillus thuringiensis 14 acatgcagca acttatttaa gtttagatgattcaggtgtt agtttaatga ataaaaatga 60 tgatgatatt gatgactata atttaaggtggtttttattt cctattgatg ataatcaata 120 tattattaca agctacgcag cgaataattgtaaggtttgg aatgttaata atgataaaat 180 aaatgtttca acttattctt caacaaactcgatacagaaa tggcaaataa aagctaatgc 240 ttcttcgtat gtaatacaaa gtaataatgggaaagttcta acagcaggaa ccggtcaatc 300 tcttggatta atacgtttaa cggatgaatcaccagataat cccaatcaac aatggaattt 360 aactcctgta caaacaattc aactcccaccaaaacctaca atagatacaa agttaaaaga 420 ttaccccaaa tattcacaaa ctggcaatatagacaaggga acacctcctc aattaatggg 480 atggacatta ataccttgta ttatggtaaatgatcccaat atagataaaa acactcaaat 540 caaaactact ccatattata ttttaaaaaaatatcaatat tggcaacaag cagtaggaag 600 taatgtagct ttacgtccgc atgaaaaaaaatcatatgct tatgagtggg gtacagaaat 660 agatcaaaaa acaactatca ttaatacattaggatttcag attaatatag attcgggaat 720 gaaatttgat ataccagaag taggtggaggtacagatgaa ataaaaacac aattaaacga 780 agaattaaaa atagaatata gccgtgaaaccaaaataatg gaaaaatat 829 15 276 PRT Bacillus thuringiensis 15 His AlaAla Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu Met 1 5 10 15 AsnLys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Arg Trp Phe Leu 20 25 30 PhePro Ile Asp Asp Asn Gln Tyr Ile Ile Thr Ser Tyr Ala Ala Asn 35 40 45 AsnCys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser Thr 50 55 60 TyrSer Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn Ala 65 70 75 80Ser Ser Tyr Val Ile Gln Ser Asn Asn Gly Lys Val Leu Thr Ala Gly 85 90 95Thr Gly Gln Ser Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Pro Asp 100 105110 Asn Pro Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln Leu 115120 125 Pro Pro Lys Pro Thr Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys Tyr130 135 140 Ser Gln Thr Gly Asn Ile Asp Lys Gly Thr Pro Pro Gln Leu MetGly 145 150 155 160 Trp Thr Leu Ile Pro Cys Ile Met Val Asn Asp Pro AsnIle Asp Lys 165 170 175 Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile LeuLys Lys Tyr Gln 180 185 190 Tyr Trp Gln Gln Ala Val Gly Ser Asn Val AlaLeu Arg Pro His Glu 195 200 205 Lys Lys Ser Tyr Ala Tyr Glu Trp Gly ThrGlu Ile Asp Gln Lys Thr 210 215 220 Thr Ile Ile Asn Thr Leu Gly Phe GlnIle Asn Ile Asp Ser Gly Met 225 230 235 240 Lys Phe Asp Ile Pro Glu ValGly Gly Gly Thr Asp Glu Ile Lys Thr 245 250 255 Gln Leu Asn Glu Glu LeuLys Ile Glu Tyr Ser Arg Glu Thr Lys Ile 260 265 270 Met Glu Lys Tyr 27516 7 PRT Bacillus thuringiensis 16 Asp Ile Asp Asp Tyr Asn Leu 1 5 17 7PRT Bacillus thuringiensis 17 Trp Phe Leu Phe Pro Ile Asp 1 5 18 8 PRTBacillus thuringiensis 18 Gln Ile Lys Thr Thr Pro Tyr Tyr 1 5 19 6 PRTBacillus thuringiensis 19 Tyr Glu Trp Gly Thr Glu 1 5 20 21 DNAArtificial Sequence Description of Artificial Sequence primer 20gatatngatg antayaaytt n 21 21 21 DNA Artificial Sequence Description ofArtificial Sequence primer 21 tggtttttnt ttccnatnga n 21 22 24 DNAArtificial Sequence Description of Artificial Sequence primer 22caaatnaaaa cnacnccata ttat 24 23 18 DNA Artificial Sequence Descriptionof Artificial Sequence primer 23 tangantggg gnacagaa 18 24 24 DNAArtificial Sequence Description of Artificial Sequence reverse primer 24ataatatggn gtngttttna tttg 24 25 18 DNA Artificial Sequence Descriptionof Artificial Sequence reverse primer 25 ttctgtnccc cantcnta 18 26 18DNA Artificial Sequence Description of Artificial Sequence forwardprimer 26 ctcaaagcgg atcaggag 18 27 20 DNA Artificial SequenceDescription of Artificial Sequence reverse primer 27 gcgtattcggatatgcttgg 20 28 386 PRT Artificial Sequence Description of ArtificialSequence generic sequence representing new class of toxins 28 Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 MetXaa Lys Xaa Asp Xaa Asp Ile Asp Asp Tyr Asn Leu Xaa Trp Phe 35 40 45 LeuPhe Pro Ile Asp Xaa Xaa Gln Tyr Ile Ile Thr Ser Tyr Xaa Ala 50 55 60 AsnAsn Cys Lys Val Trp Asn Val Xaa Asn Asp Lys Ile Asn Val Ser 65 70 75 80Thr Tyr Ser Ser Thr Asn Ser Xaa Gln Lys Trp Gln Ile Lys Ala Xaa 85 90 95Xaa Ser Ser Tyr Xaa Ile Gln Ser Xaa Asn Gly Lys Val Leu Thr Ala 100 105110 Gly Xaa Gly Gln Xaa Leu Gly Xaa Xaa Arg Leu Thr Asp Glu Xaa Xaa 115120 125 Xaa Asn Xaa Asn Gln Gln Trp Asn Leu Thr Xaa Val Gln Thr Ile Gln130 135 140 Leu Pro Xaa Lys Pro Xaa Ile Asp Xaa Lys Leu Lys Asp Xaa ProXaa 145 150 155 160 Tyr Ser Xaa Thr Gly Asn Ile Xaa Xaa Xaa Thr Xaa ProGln Leu Met 165 170 175 Gly Trp Thr Leu Xaa Pro Cys Ile Met Val Asn AspXaa Xaa Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr TyrIle Xaa Lys Lys Tyr 195 200 205 Xaa Tyr Trp Xaa Xaa Ala Xaa Gly Ser AsnVal Xaa Leu Xaa Pro His 210 215 220 Xaa Lys Xaa Ser Tyr Xaa Tyr Glu TrpGly Thr Glu Xaa Xaa Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr XaaGly Xaa Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Xaa Xaa ProGlu Val Gly Gly Gly Thr Xaa Xaa Ile Lys 260 265 270 Thr Gln Leu Xaa GluGlu Leu Lys Xaa Glu Tyr Ser Xaa Glu Thr Lys 275 280 285 Ile Met Xaa LysTyr Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 290 295 300 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 305 310 315 320 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 325 330 335Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 340 345350 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 355360 365 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa370 375 380 Xaa Xaa 385 29 28 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide probe 29 gngaagtnca tatngaaatnaataatac 28 30 2015 DNA Bacillus thuringiensis 30 attaatttta tggaggttgatatttatgtc agctcgcgaa gtacacattg aaataaacaa 60 taaaacacgt catacattacaattagagga taaaactaaa cttagcggcg gtagatggcg 120 aacatcacct acaaatgttgctcgtgatac aattaaaaca tttgtagcag aatcacatgg 180 ttttatgaca ggagtagaaggtattatata ttttagtgta aacggagacg cagaaattag 240 tttacatttt gacaatccttatataggttc taataaatgt gatggttctt ctgataaacc 300 tgaatatgaa gttattactcaaagcggatc aggagataaa tctcatgtga catatactat 360 tcagacagta tctttacgattataaggaaa atttataaaa actgtatttt ttactaaaat 420 accaaaaaat acatatttattttttggtat tttctaatat gaaatatgaa ttataaaaat 480 attaataaaa aaggtgataaaaattatgtt agatactaat aaagtttatg aaataagcaa 540 tcttgctaat ggattatatacatcaactta tttaagtctt gatgattcag gtgttagttt 600 aatgagtaaa aaggatgaagatattgatga ttacaattta aaatggtttt tatttcctat 660 tgataataat caatatattattacaagcta tggagctaat aattgtaaag tttggaatgt 720 taaaaatgat aaaataaatgtttcaactta ttcttcaaca aactctgtac aaaaatggca 780 aataaaagct aaagattcttcatatataat acaaagtgat aatggaaagg tcttaacagc 840 aggagtaggt caatctcttggaatagtacg cctaactgat gaatttccag agaattctaa 900 ccaacaatgg aatttaactcctgtacaaac aattcaactc ccacaaaaac ctaaaataga 960 tgaaaaatta aaagatcatcctgaatattc agaaaccgga aatataaatc ctaaaacaac 1020 tcctcaatta atgggatggacattagtacc ttgtattatg gtaaatgatt caaaaataga 1080 taaaaacact caaattaaaactactccata ttatattttt aaaaaatata aatactggaa 1140 tctagcaaaa ggaagtaatgtatctttact tccacatcaa aaaagatcat atgattatga 1200 atggggtaca gaaaaaaatcaaaaaacaac tattattaat acagtaggat tgcaaattaa 1260 tatagattca ggaatgaaatttgaagtacc agaagtagga ggaggtacag aagacataaa 1320 aacacaatta actgaagaattaaaagttga atatagcact gaaaccaaaa taatgacgaa 1380 atatcaagaa cactcagagatagataatcc aactaatcaa ccaatgaatt ctataggact 1440 tcttatttat acttctttagaattatatcg atataacggt acagaaatta agataatgga 1500 catagaaact tcagatcatgatacttacac tcttacttct tatccaaatc ataaagaagc 1560 attattactt ctcacaaaccattcgtatga agaagtagaa gaaataacaa aaatacctaa 1620 gcatacactt ataaaattgaaaaaacatta ttttaaaaaa taaaaaacat aatatataaa 1680 tgactgatta atatctctcgaaaaggttct ggtgcaaaaa tagtgggata tgaaaaaagc 1740 aaaagattcc taacggaatggaacattagg ctgttaaatc aaaaagttta ttgataaaat 1800 atatctgcct ttggacagacttctcccctt ggagagtttg tccttttttg accatatgca 1860 tagcttctat tccggcaatcatttttgtag ctgtttgcaa ggattttaat ccaagcatat 1920 ccgaatacgc tttttgataaccgatgtctt gttcaatgat attgtttaat attttcacac 1980 gaattggcta ctgtgcggtatcctgtctcc tttat 2015 31 360 DNA Bacillus thuringiensis 31 atgtcagctcgcgaagtaca cattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180 atatattttagtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaataaatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggagataaatctca tgtgacatat actattcaga cagtatcttt acgattataa 360 32 119 PRTBacillus thuringiensis 32 Met Ser Ala Arg Glu Val His Ile Glu Ile AsnAsn Lys Thr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys LeuSer Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg Asp ThrIle Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Val GluGly Ile Ile Tyr Phe Ser 50 55 60 Val Asn Gly Asp Ala Glu Ile Ser Leu HisPhe Asp Asn Pro Tyr Ile 65 70 75 80 Gly Ser Asn Lys Cys Asp Gly Ser SerAsp Lys Pro Glu Tyr Glu Val 85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp LysSer His Val Thr Tyr Thr Ile 100 105 110 Gln Thr Val Ser Leu Arg Leu 11533 24 DNA Artificial Sequence Description of Artificial Sequence reverseoligonucleotide primer 33 catgagattt atctcctgat ccgc 24 34 2230 DNABacillus thuringiensis 34 actatgacaa tgattatgac tgctgatgaa ttagctttatcaataccagg atattctaaa 60 ccatcaaata taacaggaga taaaagtaaa catacattatttactaatat aattggagat 120 attcaaataa aagatcaagc aacatttggg gttgtttttgatccccctct taatcgtatt 180 tcaggggctg aagaatcaag taagtttatt gatgtatattatccttctga agatagtaac 240 cttaaatatt atcaatttat aaaagtagca attgattttgatattaatga agattttatt 300 aattttaata atcatgacaa tatagggata tttaattttgttacacgaaa ttttttatta 360 aataatgaaa atgattaata aaaaatttaa tttgtataatatgtttattt tttgaaaatt 420 gaatgcatat attaatcgag tatgtgtaat aaattttaattttatggagg ttgatattta 480 tgtcagcacg tgaagtacac attgatgtaa ataataagacaggtcataca ttacaattag 540 aagataaaac aaaacttgat ggtggtagat ggcgaacatcacctacaaat gttgctaatg 600 atcaaattaa aacatttgta gcagaatcac atggttttatgacaggtaca gaaggtacta 660 tatattatag tataaatgga gaagcagaaa ttagtttatattttgacaat ccttattcag 720 gttctaataa atatgatggg cattccaata aaaatcaatatgaagttatt acccaaggag 780 gatcaggaaa tcaatctcat gttacgtata ctattcaaactgtatcttca cgatatggga 840 ataattcata aaaaaatatt tttttttacg aaaataccaaaaaaattttt ttggtatttt 900 ctaatataat tcataaatat tttaataata aaattataagaaaaggtgat aaatattatg 960 ttagatacta ataaaattta tgaaataagt aattatgctaatggattaca tgcagcaact 1020 tatttaagtt tagatgattc aggtgttagt ttaatgaataaaaatgatga tgatattgat 1080 gactataatt taaggtggtt tttatttcct attgatgataatcaatatat tattacaagc 1140 tacgcagcga ataattgtaa ggtttggaat gttaataatgataaaataaa tgtttcaact 1200 tattcttcaa caaactcgat acagaaatgg caaataaaagctaatgcttc ttcgtatgta 1260 atacaaagta ataatgggaa agttctaaca gcaggaaccggtcaatctct tggattaata 1320 cgtttaacgg atgaatcacc agataatccc aatcaacaatggaatttaac tcctgtacaa 1380 acaattcaac tcccaccaaa acctacaata gatacaaagttaaaagatta ccccaaatat 1440 tcacaaactg gcaatataga caagggaaca cctcctcaattaatgggatg gacattaata 1500 ccttgtatta tggtaaatga tccaaatata gataaaaacactcaaatcaa aactactcca 1560 tattatattt taaaaaaata tcaatattgg caacaagcagtaggaagtaa tgtagcttta 1620 cgtccgcatg aaaaaaaatc atatgcttat gagtggggtacagaaataga tcaaaaaaca 1680 actatcatta atacattagg atttcagatt aatatagattcgggaatgaa atttgatata 1740 ccagaagtag gtggaggtac agatgaaata aaaacacaattaaacgaaga attaaaaata 1800 gaatatagcc gtgaaaccaa aataatggaa aaatatcaggaacaatcaga gatagataat 1860 ccaactgatc aatcaatgaa ttctatagga ttcctcactattacttcttt agaattatat 1920 cgatataatg gttcggaaat tagtgtaatg aaaattcaaacttcagataa tgatacttac 1980 aatgtgacct cttatccaga tcatcaacaa gctctattacttcttacaaa tcattcatat 2040 gaagaagtag aagaaataac aaatattccc aaaatatcactgaaaaaatt aaaaaaatat 2100 tatttttaaa acataattat attttgatag ctttttaaaaataaagattg ttcaaagtaa 2160 aatgaaagaa aatcttttat gaaactttaa tacaataaaagaggaatatt ttcttataag 2220 tacttccttg 2230 35 372 DNA Bacillusthuringiensis 35 atgtcagcac gtgaagtaca cattgatgta aataataaga caggtcatacattacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaatgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtacagaaggtact 180 atatattata gtataaatgg agaagcagaa attagtttat attttgacaatccttattca 240 ggttctaata aatatgatgg gcattccaat aaaaatcaat atgaagttattacccaagga 300 ggatcaggaa atcaatctca tgttacgtat actattcaaa ctgtatcttcacgatatggg 360 aataattcat aa 372 36 123 PRT Bacillus thuringiensis 36Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 1015 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 2530 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 4045 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 5560 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 7075 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Asn Gln Tyr Glu Val 8590 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile100 105 110 Gln Thr Val Ser Ser Arg Tyr Gly Asn Asn Ser 115 120 37 1152DNA Bacillus thuringiensis 37 atgttagata ctaataaaat ttatgaaataagtaattatg ctaatggatt acatgcagca 60 acttatttaa gtttagatga ttcaggtgttagtttaatga ataaaaatga tgatgatatt 120 gatgactata atttaaggtg gtttttatttcctattgatg ataatcaata tattattaca 180 agctacgcag cgaataattg taaggtttggaatgttaata atgataaaat aaatgtttca 240 acttattctt caacaaactc gatacagaaatggcaaataa aagctaatgc ttcttcgtat 300 gtaatacaaa gtaataatgg gaaagttctaacagcaggaa ccggtcaatc tcttggatta 360 atacgtttaa cggatgaatc accagataatcccaatcaac aatggaattt aactcctgta 420 caaacaattc aactcccacc aaaacctacaatagatacaa agttaaaaga ttaccccaaa 480 tattcacaaa ctggcaatat agacaagggaacacctcctc aattaatggg atggacatta 540 ataccttgta ttatggtaaa tgatccaaatatagataaaa acactcaaat caaaactact 600 ccatattata ttttaaaaaa atatcaatattggcaacaag cagtaggaag taatgtagct 660 ttacgtccgc atgaaaaaaa atcatatgcttatgagtggg gtacagaaat agatcaaaaa 720 acaactatca ttaatacatt aggatttcagattaatatag attcgggaat gaaatttgat 780 ataccagaag taggtggagg tacagatgaaataaaaacac aattaaacga agaattaaaa 840 atagaatata gccgtgaaac caaaataatggaaaaatatc aggaacaatc agagatagat 900 aatccaactg atcaatcaat gaattctataggattcctca ctattacttc tttagaatta 960 tatcgatata atggttcgga aattagtgtaatgaaaattc aaacttcaga taatgatact 1020 tacaatgtga cctcttatcc agatcatcaacaagctctat tacttcttac aaatcattca 1080 tatgaagaag tagaagaaat aacaaatattcccaaaatat cactgaaaaa attaaaaaaa 1140 tattattttt aa 1152 38 383 PRTBacillus thuringiensis 38 Met Leu Asp Thr Asn Lys Ile Tyr Glu Ile SerAsn Tyr Ala Asn Gly 1 5 10 15 Leu His Ala Ala Thr Tyr Leu Ser Leu AspAsp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp AspTyr Asn Leu Arg Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asn Gln Tyr IleIle Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn AsnAsp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile GlnLys Trp Gln Ile Lys Ala Asn 85 90 95 Ala Ser Ser Tyr Val Ile Gln Ser AsnAsn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ser Leu Gly LeuIle Arg Leu Thr Asp Glu Ser Pro 115 120 125 Asp Asn Pro Asn Gln Gln TrpAsn Leu Thr Pro Val Gln Thr Ile Gln 130 135 140 Leu Pro Pro Lys Pro ThrIle Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Gln ThrGly Asn Ile Asp Lys Gly Thr Pro Pro Gln Leu Met 165 170 175 Gly Trp ThrLeu Ile Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys AsnThr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 GlnTyr Trp Gln Gln Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220Glu Lys Lys Ser Tyr Ala Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu IleLys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser Arg GluThr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp AsnPro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile ThrSer Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Ser ValMet Lys Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr SerTyr Pro Asp His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His SerTyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile Pro Lys Ile Ser LeuLys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 39 2132 DNA Bacillusthuringiensis 39 gtatttcagg gggtgaagat tcaagtaagt ttattgatgt atattatccttttgaagata 60 gtaattttaa atattatcaa tttataaaag tagcaattga ttttgatattaatgaagatt 120 ttattaattt taataatcat gacaatatag ggatatttaa ttttgttacacgaaattttt 180 tattaaataa tgaaaatgat gaataaaaaa tttaatttgt ttattatgtttattttttga 240 aaattgaatg catatattaa tcgagtatgt ataataaatt ttaattttatggaggttgat 300 atttatgtca gcacgtgaag tacacattga tgtaaataat aagacaggtcatacattaca 360 attagaagat aaaacaaaac ttgatggtgg tagatggcga acatcacctacaaatgttgc 420 taatgatcaa attaaaacat ttgtagcaga atcaaatggt tttatgacaggtacagaagg 480 tactatatat tatagtataa atggagaagc agaaattagt ttatattttgacaatccttt 540 tgcaggttct aataaatatg atggacattc caataaatct caatatgaaattattaccca 600 aggaggatca ggaaatcaat ctcatgttac gtatactatt caaaccacatcctcacgata 660 tgggcataaa tcataacaaa taatttttta cgaaaatacc aaaaaataaatattttttgg 720 tattttctaa tataaattac aaatatatta ataataaaat tataagaaaaggtgataaag 780 attatgttag atactaataa agtttatgaa ataagcaatc atgctaatggactatatgca 840 gcaacttatt taagtttaga tgattcaggt gttagtttaa tgaataaaaatgatgatgat 900 attgatgatt ataacttaaa atggttttta tttcctattg atgatgatcaatatattatt 960 acaagctatg cagcaaataa ttgtaaagtt tggaatgtta ataatgataaaataaatgtt 1020 tcgacttatt cttcaacaaa ttcaatacaa aaatggcaaa taaaagctaatggttcttca 1080 tatgtaatac aaagtgataa tggaaaagtc ttaacagcag gaaccggtcaagctcttgga 1140 ttgatacgtt taactgatga atcctcaaat aatcccaatc aacaatggaatttaacttct 1200 gtacaaacaa ttcaacttcc acaaaaacct ataatagata caaaattaaaagattatccc 1260 aaatattcac caactggaaa tatagataat ggaacatctc ctcaattaatgggatggaca 1320 ttagtacctt gtattatggt aaatgatcca aatatagata aaaatactcaaattaaaact 1380 actccatatt atattttaaa aaaatatcaa tattggcaac gagcagtaggaagtaatgta 1440 gctttacgtc cacatgaaaa aaaatcatat acttatgaat ggggcacagaaatagatcaa 1500 aaaacaacaa ttataaatac attaggattt caaatcaata tagattcaggaatgaaattt 1560 gatataccag aagtaggtgg aggtacagat gaaataaaaa cacaactaaatgaagaatta 1620 aaaatagaat atagtcatga aactaaaata atggaaaaat atcaagaacaatctgaaata 1680 gataatccaa ctgatcaatc aatgaattct ataggatttc ttactattacttccttagaa 1740 ttatatagat ataatggctc agaaattcgt ataatgcaaa ttcaaacctcagataatgat 1800 acttataatg ttacttctta tccaaatcat caacaagctt tattacttcttacaaatcat 1860 tcatatgaag aagtagaaga aataacaaat attcctaaaa gtacactaaaaaaattaaaa 1920 aaatattatt tttaaatatt gaaattagaa attatctaaa acaaaacgaaagataattta 1980 atctttaatt atttgtaaga taatcgtatt ttatttgtat taatttttatacaatataaa 2040 gtaatatctg tacgtgaaat tggtttcgct tcaatatcta atctcatctcatgtattaca 2100 tgcgtaatac cttcttgttc tgcttctaca ag 2132 40 372 DNABacillus thuringiensis 40 atgtcagcac gtgaagtaca cattgatgta aataataagacaggtcatac attacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacatcacctacaaa tgttgctaat 120 gatcaaatta aaacatttgt agcagaatca aatggttttatgacaggtac agaaggtact 180 atatattata gtataaatgg agaagcagaa attagtttatattttgacaa tccttttgca 240 ggttctaata aatatgatgg acattccaat aaatctcaatatgaaattat tacccaagga 300 ggatcaggaa atcaatctca tgttacgtat actattcaaaccacatcctc acgatatggg 360 cataaatcat aa 372 41 123 PRT Bacillusthuringiensis 41 Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys ThrGly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly GlyArg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys ThrPhe Val Ala 35 40 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr IleTyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp AsnPro Phe Ala 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys SerGln Tyr Glu Ile 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His ValThr Tyr Thr Ile 100 105 110 Gln Thr Thr Ser Ser Arg Tyr Gly His Lys Ser115 120 42 1241 DNA Bacillus thuringiensis misc_feature (18)Undetermined nucleotide 42 wcdmtkdvrm wahkcmdndb ygtrawbmkg cwtkctgyhdcywagmawtd cvnwmhasrt 60 nchhtmsnwr manrgarcrr nwrgarhatg ttagatactaataaagttta tgaaataagc 120 aatcatgcta atggactata tgcagcaact tatttaagtttagatgattc aggtgttagt 180 ttaatgaata aaaatgatga tgatattgat gattataacttaaaatggtt tttatttcct 240 attgatgatg atcaatatat tattacaagc tatgcagcaaataattgtaa agtttggaat 300 gttaataatg ataaaataaa tgtttcgact tattcttcaacaaattcaat acaaaaatgg 360 caaataaaag ctaatggttc ttcatatgta atacaaagtgataatggaaa agtcttaaca 420 gcaggaaccg gtcaagctct tggattgata cgtttaactgatgaatcctc aaataatccc 480 aatcaacaat ggaatttaac ttctgtacaa acaattcaacttccacaaaa acctataata 540 gatacaaaat taaaagatta tcccaaatat tcaccaactggaaatataga taatggaaca 600 tctcctcaat taatgggatg gacattagta ccttgtattatggtaaatga tccaaatata 660 gataaaaata ctcaaattaa aactactcca tattatattttaaaaaaata tcaatattgg 720 caacgagcag taggaagtaa tgtagcttta cgtccacatgaaaaaaaatc atatacttat 780 gaatggggca cagaaataga tcaaaaaaca acaattataaatacattagg atttcaaatc 840 aatatagatt caggaatgaa atttgatata ccagaagtaggtggaggtac agatgaaata 900 aaaacacaac taaatgaaga attaaaaata gaatatagtcatgaaactaa aataatggaa 960 aaatatcaag aacaatctga aatagataat ccaactgatcaatcaatgaa ttctatagga 1020 tttcttacta ttacttcctt agaattatat agatataatggctcagaaat tcgtataatg 1080 caaattcaaa cctcagataa tgatacttat aatgttacttcttatccaaa tcatcaacaa 1140 gctttattac ttcttacaaa tcattcatat gaagaagtagaagaaataac aaatattcct 1200 aaaagtacac taaaaaaatt aaaaaaatat tatttttaav v1241 43 383 PRT Bacillus thuringiensis 43 Met Leu Asp Thr Asn Lys ValTyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr TyrLeu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp AspAsp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp AspAsp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val TrpAsn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser ThrAsn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr ValIle Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly GlnAla Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn ProAsn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu ProGln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg ProHis 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile AspGln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile AsnIle Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly GlyThr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile GluTyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln SerGlu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn Ser Ile Gly PheLeu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly SerGlu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr TyrAsn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 Leu Leu Leu LeuThr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365 Asn Ile ProLys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe 370 375 380 44 360 DNAArtificial Sequence Description of Artificial Sequence maize-optimizedgene sequence 44 atgtccgccc gcgaggtgca catcgagatc aacaacaaga cccgccacaccctccagctc 60 gaggacaaga ccaagctctc cggcggcagg tggcgcacct ccccgaccaacgtggcccgc 120 gacaccatca agacgttcgt ggcggagtcc cacggcttca tgaccggcgtcgagggcatc 180 atctacttct ccgtgaacgg cgacgccgag atctccctcc acttcgacaacccgtacatc 240 ggctccaaca agtgcgacgg ctcctccgac aagcccgagt acgaggtgatcacccagtcc 300 ggctccggcg acaagtccca cgtgacctac accatccaga ccgtgtccctccgcctctga 360 45 1158 DNA Artificial Sequence Description of ArtificialSequence maize-optimized gene sequence 45 atgctcgaca ccaacaaggtgtacgagatc tccaacctcg ccaacggcct ctacacctcc 60 acctacctct ccctcgacgactccggcgtg tccctcatgt ccaagaagga cgaggacatc 120 gacgactaca acctcaagtggttcctcttc ccgatcgaca acaaccagta catcatcacc 180 tcctacggcg ccaacaactgcaaggtgtgg aacgtgaaga acgacaagat caacgtgtcc 240 acctactcct ccaccaactccgtgcagaag tggcagatca aggccaagga ctcctcctac 300 atcatccagt ccgacaacggcaaggtgctc accgcgggcg tgggccagtc cctcggcatc 360 gtgcgcctca ccgacgagttcccggagaac tccaaccagc aatggaacct caccccggtg 420 cagaccatcc agctcccgcagaagccgaag atcgacgaga agctcaagga ccacccggag 480 tactccgaga ccggcaacatcaacccgaag accaccccgc agctcatggg ctggaccctc 540 gtgccgtgca tcatggtgaacgactccaag atcgacaaga acacccagat caagaccacc 600 ccgtactaca tcttcaagaaatacaagtac tggaacctcg ccaagggctc caacgtgtcc 660 ctcctcccgc accagaagcgcagctacgac tacgagtggg gcaccgagaa gaaccagaag 720 accaccatca tcaacaccgtgggcctgcag atcaacatcg actcggggat gaagttcgag 780 gtgccggagg tgggcggcggcaccgaggac atcaagaccc agctcaccga ggagctgaag 840 gtggagtact ccaccgagaccaagatcatg accaagtacc aggagcactc cgagatcgac 900 aacccgacca accagccgatgaactccatc ggcctcctca tctacacctc cctcgagctg 960 taccgctaca acggcaccgagatcaagatc atggacatcg agacctccga ccacgacacc 1020 tacaccctca cctcctacccgaaccacaag gaggcgctgc tgctgctgac caaccactcc 1080 tacgaggagg tggaggagatcaccaagatc ccgaagcaca ccctcatcaa gctcaagaag 1140 cactacttca agaagtga1158 46 24 DNA Artificial Sequence Description of Artificial Sequencereverse primer 46 gtagaagcag aacaagaagg tatt 24 47 25 DNA ArtificialSequence Description of Artificial Sequence forward primer 47 atgtcagcwcgygaagtwca yattg 25 48 23 DNA Artificial Sequence Description ofArtificial Sequence reverse primer 48 gtytgaathg tatahgthac atg 23 49 25DNA Artificial Sequence Description of Artificial Sequence forwardprimer 49 atgttagata cwaataaart wtatg 25 50 29 DNA Artificial SequenceDescription of Artificial Sequence reverse primer 50 gtwatttcttcwacttcttc atahgaatg 29 51 341 DNA Bacillus thuringiensis 51 atgtcaggtcgagaagtaca tattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180 atatattttagtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaataaatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggagataaatctca tgtaacatat actattcaga c 341 52 113 PRT Bacillus thuringiensis52 Met Ser Gly Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 5055 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 6570 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile100 105 110 Gln 53 1103 DNA Bacillus thuringiensis 53 atgttagatacaaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatcm 60 acttatttaagtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120 gatgattacaatttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180 agctatggagctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240 acttattcttcaacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300 ataatacaaagtgataatgg aaaggtctta acagcaggag taggtcaatc tcttggaata 360 gtacgcctaactgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420 caaacaattcaactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480 tattcagaaaccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540 gtaccttgtattatggtaaa tgattcaaaa atagataaaa acactcaaat taaaactact 600 ccatattatatttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660 ttacttccacatcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720 acamctattattaatacagt aggattgcaa attaatatag actcaggaat gaaatttgaa 780 gtaccagaagtaggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840 gttgaatatagcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900 aatccaactaatcaaccaat gaattctata ggacttctta tttacacttc tttagaatta 960 tatcgatataacggtacaga aattaagata atggacatag aaacttcaga tcatgatact 1020 tacactcttacttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattca 1080 tatgaagaagtagaagaaat aac 1103 54 367 PRT Bacillus thuringiensis MISC_FEATURE (242)Undetermined amino acid 54 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile SerAsn Leu Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu AspAsp Ser Gly Val Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile Asp AspTyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr IleIle Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys AsnAsp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Val GlnLys Trp Gln Ile Lys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln Ser AspAsn Gly Lys Val Leu Thr Ala 100 105 110 Gly Val Gly Gln Ser Leu Gly IleVal Arg Leu Thr Asp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln Gln TrpAsn Leu Thr Pro Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro LysIle Asp Glu Lys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser Glu ThrGly Asn Ile Asn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly Trp ThrLeu Val Pro Cys Ile Met Val Asn Asp Ser Lys Ile Asp 180 185 190 Lys AsnThr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205 LysTyr Trp Asn Leu Ala Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220Gln Lys Arg Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230235 240 Thr Xaa Ile Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp IleLys 260 265 270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr GluThr Lys 275 280 285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile Asp AsnPro Thr Asn 290 295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile Tyr ThrSer Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Thr Glu Ile Lys IleMet Asp Ile Glu Thr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr SerTyr Pro Asn His Lys Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn His SerTyr Glu Glu Val Glu Glu Ile 355 360 365 55 341 DNA Bacillusthuringiensis 55 atgtcagctc gtgaagtaca tattgatgta aataataaga caggtcatacattacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaatgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtacagaaggtcat 180 atatattata gtataaatgg agaagcagaa attagtttat attttgataatccttattca 240 ggttctaata aatatgatgg ggattccaat aaacctcaat atgaagttactacccaagga 300 ggatcaggaa atcaatctca tgtaacatat acgattcaaa c 341 56 113PRT Bacillus thuringiensis 56 Met Ser Ala Arg Glu Val His Ile Asp ValAsn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr LysLeu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn AspGln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly ThrGlu Gly His Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser LeuTyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly AspSer Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly AsnGln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 57 1103 DNA Bacillusthuringiensis misc_feature (1028) Undetermined nucleotide 57 atgttagatactaataaagt ttatgaaata agtaatcatg ctaatggact atatgcagca 60 acttatttaagtttagatga ttcaggtgtt agtttaatga ataaaaatga tgatgatatt 120 gatgattacaacttaaaatg gtttttattt cctattgatg atgatcaata tattattaca 180 agctatgcagcaaataattg taaagtttgg aatgttaata atgataaaat aaatgtttcg 240 acttattctttaacaaattc aatacaaaaa tggcaaataa aagctaatgg ttcttcatat 300 gtaatacaaagtgataatgg aaaagtctta acagcaggaa ccggtcaagc tcttggattg 360 atacgtttaactgatgaatc ttcaaataat cccaatcaac aatggaattt aacttctgta 420 caaacaattcaacttccaca aaaacctata atagatacaa aattaaaaga ttatcccaaa 480 tattcaccaactggaaatat agataatgga acatctcctc aattaatggg atggacatta 540 gtaccttgtattatggtaaa tgatccaaat atagataaaa atactcaaat taaaactact 600 ccatattatattttaaaaaa atatcaatat tggcaacgag cagtaggaag taatgtagct 660 ttacgtccacatgaaaaaaa atcatatact tatgaatggg gaacagaaat agatcaaaaa 720 acaacaatcataaatacatt aggatttcaa atcaatatag attcaggaat gaaatttgat 780 ataccagaagtaggtggagg tacagatgaa ataaaaacac aactaaatga agaattaaaa 840 atagaatatagtcgtgaaac taaaataatg gaaaaatatc aagaacaatc tgaaatagat 900 aatccaactgatcaaccaat gaattctata ggatttctta ctattacttc tttagaatta 960 tatagatataatggctcaga aattcgtata atgcaaattc aaacctcaga taatgatact 1020 tataatgntacttcttatcc agatcatcaa caagctttat tacttcttac aaatcattca 1080 tatgaagaactagaagaaat aac 1103 58 367 PRT Bacillus thuringiensis MISC_FEATURE (343)Undetermined amino acid 58 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile SerAsn His Ala Asn Gly 1 5 10 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu AspAsp Ser Gly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp AspTyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr IleIle Thr Ser Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn AsnAsp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Leu Thr Asn Ser Ile GlnLys Trp Gln Ile Lys Ala Asn 85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser AspAsn Gly Lys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ala Leu Gly LeuIle Arg Leu Thr Asp Glu Ser Ser 115 120 125 Asn Asn Pro Asn Gln Gln TrpAsn Leu Thr Ser Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro IleIle Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Pro ThrGly Asn Ile Asp Asn Gly Thr Ser Pro Gln Leu Met 165 170 175 Gly Trp ThrLeu Val Pro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys AsnThr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 GlnTyr Trp Gln Arg Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230235 240 Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly245 250 255 Met Lys Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu IleLys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser Arg GluThr Lys 275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp AsnPro Thr Asp 290 295 300 Gln Pro Met Asn Ser Ile Gly Phe Leu Thr Ile ThrSer Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg IleMet Gln Ile Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Xaa Thr SerTyr Pro Asp His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His SerTyr Glu Glu Leu Glu Glu Ile 355 360 365 59 340 DNA Bacillusthuringiensis 59 atgtcagcag gtgaagtaca tattgatgca aataataaga caggtcatacattacaatta 60 gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaatgttgctaat 120 gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtacagaaggtcat 180 atatattata gtataaatgg agaagcagaa attagtttat attttgataatccttattca 240 ggttctaata aatatgatgg ggattccaat aaacctcaat atgaagttactacccaagga 300 ggatcaggaa atcaatctca tgttacttat acaattcaaa 340 60 113PRT Bacillus thuringiensis 60 Met Ser Ala Gly Glu Val His Ile Asp AlaAsn Asn Lys Thr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr LysLeu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn AspGln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly ThrGlu Gly His Ile Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser LeuTyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly AspSer Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly AsnGln Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 61 340 DNA Bacillusthuringiensis 61 tgtcagcacg tgaagtacat attgaaataa acaataaaac acgtcatacattacaattag 60 aggataaaac taaacttagc ggcggtagat ggcgaacatc acctacaaatgttgctcgtg 120 atacaattaa aacatttgta gcagaatcac atggttttat gacaggagtagaaggtatta 180 tatattttag tgtaaacgga gacgcagaaa ttagtttaca ttttgacaatccttatatag 240 gttctaataa atgtgatggt tcttctgata aacctgaata tgaagttattactcaaagcg 300 gatcaggaga taaatctcat gtgacatata cgattcagac 340 62 112PRT Bacillus thuringiensis 62 Ser Ala Arg Glu Val His Ile Glu Ile AsnAsn Lys Thr Arg His Thr 1 5 10 15 Leu Gln Leu Glu Asp Lys Thr Lys LeuSer Gly Gly Arg Trp Arg Thr 20 25 30 Ser Pro Thr Asn Val Ala Arg Asp ThrIle Lys Thr Phe Val Ala Glu 35 40 45 Ser His Gly Phe Met Thr Gly Val GluGly Ile Ile Tyr Phe Ser Val 50 55 60 Asn Gly Asp Ala Glu Ile Ser Leu HisPhe Asp Asn Pro Tyr Ile Gly 65 70 75 80 Ser Asn Lys Cys Asp Gly Ser SerAsp Lys Pro Glu Tyr Glu Val Ile 85 90 95 Thr Gln Ser Gly Ser Gly Asp LysSer His Val Thr Tyr Thr Ile Gln 100 105 110 63 1114 DNA Bacillusthuringiensis 63 atgttagata ctaataaaat ttatgaaata agcaatcttg ctaatggattatatacatca 60 acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaaggatgaagatatt 120 gatgattaca atttaaaatg gtttttattt cctattgata ataatcaatatattattaca 180 agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaataaatgtttca 240 acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaagattcttcatat 300 ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatctcttggaata 360 gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaatttaactcctgta 420 caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaagatcatcctgaa 480 tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatgggatggacatta 540 gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaattaaaactact 600 ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaagtaatgtatct 660 ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaaaaatcaaaaa 720 acaactatta ttaatacagt aggattgcaa attaatatag attcaggaatgaaatttgaa 780 gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactgaagaattaaaa 840 gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactcagagatagat 900 aatccaacta atcaaccaat gaattctata ggacttctta tttatacttctttagaatta 960 tatcgatata acggtacaga aattaagata atggacatag aaacttcagatcatgatact 1020 tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcacaaaccattct 1080 tatgaagaac tagaacaaat tacaagggcg aatt 1114 64 371 PRTBacillus thuringiensis 64 Met Leu Asp Thr Asn Lys Ile Tyr Glu Ile SerAsn Leu Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu AspAsp Ser Gly Val Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile Asp AspTyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr IleIle Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys AsnAsp Lys Ile Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Val GlnLys Trp Gln Ile Lys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln Ser AspAsn Gly Lys Val Leu Thr Ala 100 105 110 Gly Val Gly Gln Ser Leu Gly IleVal Arg Leu Thr Asp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln Gln TrpAsn Leu Thr Pro Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro LysIle Asp Glu Lys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser Glu ThrGly Asn Ile Asn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly Trp ThrLeu Val Pro Cys Ile Met Val Asn Asp Ser Lys Ile Asp 180 185 190 Lys AsnThr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205 LysTyr Trp Asn Leu Ala Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220Gln Lys Arg Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230235 240 Thr Thr Ile Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp IleLys 260 265 270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr GluThr Lys 275 280 285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile Asp AsnPro Thr Asn 290 295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile Tyr ThrSer Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Thr Glu Ile Lys IleMet Asp Ile Glu Thr Ser 325 330 335 Asp His Asp Thr Tyr Thr Leu Thr SerTyr Pro Asn His Lys Glu Ala 340 345 350 Leu Leu Leu Leu Thr Asn His SerTyr Glu Glu Leu Glu Gln Ile Thr 355 360 365 Arg Ala Asn 370 65 360 DNABacillus thuringiensis 65 atgtcagctc gcgaagtaca cattgaaata aacaataaaacacgtcatac attacaatta 60 gaggataaaa ctaaacttag cggcggtaga tggcgaacatcacctacaaa tgttgctcgt 120 gatacaatta aaacatttgt agcagaatca catggttttatgacaggagt agaaggtatt 180 atatatttta gtgtaaacgg agacgcagaa attagtttacattttgacaa tccttatata 240 ggttctaata aatgtgatgg ttcttctgat aaacctgaatatgaagttat tactcaaagc 300 ggatcaggag ataaatctca tgtgacatat actattcagacagtatcttt acgattataa 360 66 119 PRT Bacillus thuringiensis 66 Met SerAla Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15 ThrLeu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30 ThrSer Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45 GluSer His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 50 55 60 ValAsn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 65 70 75 80Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile 100 105110 Gln Thr Val Ser Leu Arg Leu 115 67 1158 DNA Bacillus thuringiensis67 atgttagata ctaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatca 60acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120gatgattaca atttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtcaatc tcttggaata 360gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgattcaaaa atagataaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720acaactatta ttaatacagt aggattgcaa attaatatag attcaggaat gaaatttgaa 780gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900aatccaacta atcaaccaat gaattctata ggacttctta tttatacttc tttagaatta 960tatcgatata acggtacaga aattaagata atggacatag aaacttcaga tcatgatact 1020tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattcg 1080tatgaagaag tagaagaaat aacaaaaata cctaagcata cacttataaa attgaaaaaa 1140cattatttta aaaaataa 1158 68 385 PRT Bacillus thuringiensis 68 Met LeuAsp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala Asn Gly 1 5 10 15 LeuTyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 MetSer Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 LeuPhe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 AsnAsn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Ile Asn Val Ser 65 70 75 80Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Phe Pro 115120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Glu Lys Leu Lys Asp His ProGlu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Asn Pro Lys Thr Thr ProGln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn AspSer Lys Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr TyrIle Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn Leu Ala Lys Gly Ser AsnVal Ser Leu Leu Pro His 210 215 220 Gln Lys Arg Ser Tyr Asp Tyr Glu TrpGly Thr Glu Lys Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr ValGly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Glu Val ProGlu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265 270 Thr Gln Leu Thr GluGlu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280 285 Ile Met Thr LysTyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Pro MetAsn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu Glu Leu 305 310 315 320 TyrArg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp Ile Glu Thr Ser 325 330 335Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Lys Glu Ala 340 345350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355360 365 Lys Ile Pro Lys His Thr Leu Ile Lys Leu Lys Lys His Tyr Phe Lys370 375 380 Lys 385 69 341 DNA Bacillus thuringiensis 69 atgtcagcacgagaagtaca cattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatctgt agcagaatca aatggtttta tgacaggtac agaaggtact 180 atatattatagtataaatgg agaagcagaa attagtttat attttgacaa tccttttgca 240 ggttctaataaatatgatgg acattccaat aaatctcaat atgaaattat tacccaagga 300 ggatcaggaaatcaatctca tgttacttat acaattcaga c 341 70 113 PRT Bacillus thuringiensis70 Met Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Ser Val Ala 3540 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 5055 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile100 105 110 Gln 71 340 DNA Bacillus thuringiensis 71 atgtcagcaggcgaagttca tattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatttgt agcagaatca aatggtttta tgacaggtac agaaggtact 180 atatattatagtataaatgg agaagcagaa attagtttat attttgacaa tccttttgca 240 ggttctaataaatatgatgg acattccaat aaatctcaat atgaaattat tacccaagga 300 ggatcaggaaatcaatctca tgtaacgtat acaattcaaa 340 72 113 PRT Bacillus thuringiensis72 Met Ser Ala Gly Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 3540 45 Glu Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 5055 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile100 105 110 Gln 73 340 DNA Bacillus thuringiensis 73 atgtcagctcgcgaagtwca tattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180 atatattttagtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaataaatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggagataaatctca tgtgacatat accattcaaa 340 74 113 PRT Bacillus thuringiensis74 Met Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 5055 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 6570 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile100 105 110 Gln 75 341 DNA Bacillus thuringiensis 75 atgtcagctcgcgaagttca tattgaaata aataataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttac cagtggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggaat agaaggtatt 180 atatattttagcgtaaacgg agaagcagaa attagtttac attttgacaa tccttatgta 240 ggttctaataaatatgatgg ttcttctgat aaagctgcat acgaagttat tgctcaaggt 300 ggatcaggggatatatctca tgtaacttat acaattcaaa c 341 76 113 PRT Bacillus thuringiensis76 Met Ser Ala Arg Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Thr Ser Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Ile Glu Gly Ile Ile Tyr Phe Ser 5055 60 Val Asn Gly Glu Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Val 6570 75 80 Gly Ser Asn Lys Tyr Asp Gly Ser Ser Asp Lys Ala Ala Tyr Glu Val85 90 95 Ile Ala Gln Gly Gly Ser Gly Asp Ile Ser His Val Thr Tyr Thr Ile100 105 110 Gln 77 1175 DNA Bacillus thuringiensis 77 atgttagatactaataaagt ttatgaaata agcaatcatg ctaatggatt atatacatca 60 acttatttaagtctggatga ttcaggtgtt agtttaatgg gtcaaaatga tgaggatata 120 gatgaatmcaatttaaagtg gttcttattt ccaatagata ataatcaata tattattaca 180 agctatggagcgaataattg taaagtttgg aatgttaaaa atgataaagt aaatgtttca 240 acgtattctccaacaaactc agtacaaaaa tggcaaataa aagctaaaaa ttcttcatat 300 ataatacaaagtgagaatgg aaaagtctta acagcaggaa taggtcaatc tcctggaata 360 gtacgcttaaccgatgaatc atcagagagt tctaaccaac aatggaattt aatccctgta 420 caaacaatttcactcccaca aaaacctaaa atagataaaa aattaaaaga tcatcctgaa 480 tattcagaaaccggaaatat agctactgga acaattcctc aattaatggg atggacatta 540 gtaccttgtattatggtaaa tgatccaaaa atagataaaa acactcaaat taaaactact 600 ccatattatatttttaaaaa atatcaatac tggaaacgag caataggaag taatgtatct 660 ttacttccacatcaaaaaaa atcatatgat tatgagtggg gtacagaaga aaatcaaaaa 720 acaactattattaatacagt aggatttcaa attaatgtag attcaggaat gaagtttgag 780 gtaccagaagtaggaggagg tacagaagaa ataaaaacac aattaaatga agaattaaaa 840 gttgaatatagcactgacac caaaataatg aaaaaatatc aagaacactc agagatagat 900 aatccaactaatcaaacaat gaattctata ggatttctta cttttacttc tttagaatta 960 tatcgatataacggttcgga aattcgtata atgagaatgg aaacttcaga taatgatact 1020 tatactctgacctcttatcc aaatcataga gaagcattat tacttctcac aaatcattca 1080 tatcaagaagtacmagaaat tacaagggcg aattcttgca gatatccatc acactggcgg 1140 gccggtcgagccttgcatct agaggggccc caatt 1175 78 391 PRT Bacillus thuringiensisMISC_FEATURE (43) Undetermined amino acid 78 Met Leu Asp Thr Asn Lys ValTyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser Thr TyrLeu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Gly Gln Asn Asp GluAsp Ile Asp Glu Xaa Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp AsnAsn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys Val TrpAsn Val Lys Asn Asp Lys Val Asn Val Ser 65 70 75 80 Thr Tyr Ser Pro ThrAsn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95 Asn Ser Ser Tyr IleIle Gln Ser Glu Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Ile Gly GlnSer Pro Gly Ile Val Arg Leu Thr Asp Glu Ser Ser 115 120 125 Glu Ser SerAsn Gln Gln Trp Asn Leu Ile Pro Val Gln Thr Ile Ser 130 135 140 Leu ProGln Lys Pro Lys Ile Asp Lys Lys Leu Lys Asp His Pro Glu 145 150 155 160Tyr Ser Glu Thr Gly Asn Ile Ala Thr Gly Thr Ile Pro Gln Leu Met 165 170175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Lys Ile Asp 180185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr195 200 205 Gln Tyr Trp Lys Arg Ala Ile Gly Ser Asn Val Ser Leu Leu ProHis 210 215 220 Gln Lys Lys Ser Tyr Asp Tyr Glu Trp Gly Thr Glu Glu AsnGln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Val Gly Phe Gln Ile AsnVal Asp Ser Gly 245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly Gly GlyThr Glu Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys Val GluTyr Ser Thr Asp Thr Lys 275 280 285 Ile Met Lys Lys Tyr Gln Glu His SerGlu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Thr Met Asn Ser Ile Gly PheLeu Thr Phe Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly SerGlu Ile Arg Ile Met Arg Met Glu Thr Ser 325 330 335 Asp Asn Asp Thr TyrThr Leu Thr Ser Tyr Pro Asn His Arg Glu Ala 340 345 350 Leu Leu Leu LeuThr Asn His Ser Tyr Gln Glu Val Xaa Glu Ile Thr 355 360 365 Arg Ala AsnSer Cys Arg Tyr Pro Ser His Trp Arg Ala Gly Arg Ala 370 375 380 Leu HisLeu Glu Gly Pro Gln 385 390 79 341 DNA Bacillus thuringiensis 79atgtcagcag gtgaagttca tattgaaata aataataaaa cacgtcatac attacaatta 60gaggataaaa ctaaacttac cagtggtaga tggcgaacat cacctacaaa tgttgctcgt 120gatacaatta aaacatttgt agcagaatca catggtttta tgacaggaat agaaggtatt 180atatatttta gcgtaaacgg agaagcagaa attagtttac attttgacaa tccttatgta 240ggttctaata aatatgatgg ttcttctgat aaagctgcat acgaagttat tgctcaaggt 300ggatcagggg atatatctca tctaacatat acaattcaaa c 341 80 113 PRT Bacillusthuringiensis 80 Met Ser Ala Gly Glu Val His Ile Glu Ile Asn Asn Lys ThrArg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Thr Ser GlyArg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys ThrPhe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Ile Glu Gly Ile IleTyr Phe Ser 50 55 60 Val Asn Gly Glu Ala Glu Ile Ser Leu His Phe Asp AsnPro Tyr Val 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly Ser Ser Asp Lys AlaAla Tyr Glu Val 85 90 95 Ile Ala Gln Gly Gly Ser Gly Asp Ile Ser His LeuThr Tyr Thr Ile 100 105 110 Gln 81 1410 DNA Bacillus thuringiensis 81atgttagata ctaataaaat ttatgaaata agcaatcatg ctaatggatt atatacatca 60acttatttaa gtctggatga ttcaggtgtt agtttaatgg gtcaaaatga tgaggatata 120gatgaataca atttaaagtg gttcttattt ccaatagata ataatcaata tattattaca 180agctatggag cgaataattg taaagtttgg aatgttaaaa atgataaagt aaatgtttca 240acgtattctc caacaaactc agtacaaaaa tggcaaataa aagctaaaaa ttcttcatat 300ataatacaaa gtgagaatgg aaaagtctta acagcaggaa taggtcaatc tcttggaata 360gtacgcttaa ccgatgaatc atcagagagt tctaaccaac aatggaattt aatccctgta 420caaacaattt cactcccaca aaaacctaaa atagataaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat agctactgga acaattcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgatccaaaa ataggtaaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atatcaatac tggaaacgag caataggaag taatgtatct 660ttacttccac atcaaaaaaa atcatatgat tatgagtggg gtacagaaga aaatcaaaaa 720acaactatta ttaatacagt aggatttcaa attaatgtag attcaggaat gaagtttgag 780gtaccagaag taggaggagg tacagaagaa ataaaaacac aattaaatga agaattaaaa 840gttgaatata gcactgacac caaaataatg aaaaaatatc aagaacactc agagatagat 900aatccaacta atcaaacaac gaattctata ggatttctta cttttacttc tttagaatta 960tatcgatata acggttcgga aattcgtata atgagaatgg aaacttcaga taatgatact 1020tatactctga cctcttatcc aaatcataga gaagcattat tacttctcac aaatcattct 1080tatcaagaag taagccgaat tccagcacac tggcggccgt tactagtgga tccgagctcg 1140gtaccaagct tggcgtaatc atggtcatag stgtttcctg tgtgaaattg ttatccgctc 1200acaattccac acaacatacg agccggaagc ataaagtgta aagcctgggg tgcctaatga 1260gtgagctaac tcacattaat tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg 1320tcgtgccagc tgcattaatg aatcggccaa cgcgcgggga gaggcggttt gcgtattggg 1380cgctcttccg cttcctcgct cactgactcg 1410 82 462 PRT Bacillus thuringiensisMISC_FEATURE (389) Undetermined amino acid 82 Met Leu Asp Thr Asn LysIle Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Thr Ser ThrTyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Gly Gln Asn AspGlu Asp Ile Asp Glu Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile AspAsn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys Lys ValTrp Asn Val Lys Asn Asp Lys Val Asn Val Ser 65 70 75 80 Thr Tyr Ser ProThr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95 Asn Ser Ser TyrIle Ile Gln Ser Glu Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly Ile GlyGln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Ser Ser 115 120 125 Glu SerSer Asn Gln Gln Trp Asn Leu Ile Pro Val Gln Thr Ile Ser 130 135 140 LeuPro Gln Lys Pro Lys Ile Asp Lys Lys Leu Lys Asp His Pro Glu 145 150 155160 Tyr Ser Glu Thr Gly Asn Ile Ala Thr Gly Thr Ile Pro Gln Leu Met 165170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Lys Ile Gly180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe Lys LysTyr 195 200 205 Gln Tyr Trp Lys Arg Ala Ile Gly Ser Asn Val Ser Leu LeuPro His 210 215 220 Gln Lys Lys Ser Tyr Asp Tyr Glu Trp Gly Thr Glu GluAsn Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Val Gly Phe Gln IleAsn Val Asp Ser Gly 245 250 255 Met Lys Phe Glu Val Pro Glu Val Gly GlyGly Thr Glu Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu Lys ValGlu Tyr Ser Thr Asp Thr Lys 275 280 285 Ile Met Lys Lys Tyr Gln Glu HisSer Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Thr Thr Asn Ser Ile GlyPhe Leu Thr Phe Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr Asn GlySer Glu Ile Arg Ile Met Arg Met Glu Thr Ser 325 330 335 Asp Asn Asp ThrTyr Thr Leu Thr Ser Tyr Pro Asn His Arg Glu Ala 340 345 350 Leu Leu LeuLeu Thr Asn His Ser Tyr Gln Glu Val Ser Arg Ile Pro 355 360 365 Ala HisTrp Arg Pro Leu Leu Val Asp Pro Ser Ser Val Pro Ser Leu 370 375 380 AlaSer Trp Ser Xaa Phe Pro Val Asn Cys Tyr Pro Leu Thr Ile Pro 385 390 395400 His Asn Ile Arg Ala Gly Ser Ile Lys Cys Lys Ala Trp Gly Ala Val 405410 415 Ser Leu Thr Leu Ile Ala Leu Arg Ser Leu Pro Ala Phe Gln Ser Gly420 425 430 Asn Leu Ser Cys Gln Leu His Ile Gly Gln Arg Ala Gly Arg GlyGly 435 440 445 Leu Arg Ile Gly Arg Ser Ser Ala Ser Ser Leu Thr Asp Ser450 455 460 83 340 DNA Bacillus thuringiensis 83 tgtcagcacg tgaagtacatattgatgtaa ataataagac aggtcataca ttacaattag 60 aagataaaac aaaacttgatggtggtagat ggcgaacatc acctacaaat gttgctaatg 120 atcaaattaa aacatttgtagcagaatcaa atggttttat gacaggtaca gaaggtacta 180 tatattatag tataaatggagaagcagaaa ttagtttata ttttgacaat ccttttgcag 240 gttctaataa atatgatggacattccaata aatctcaata tgaaattatt acccaaggag 300 gatcaggaaa tcaatctcatgtgacatata ctattcaaac 340 84 112 PRT Bacillus thuringiensis 84 Ser AlaArg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 1 5 10 15 LeuGln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg Thr 20 25 30 SerPro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala Glu 35 40 45 SerAsn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser Ile 50 55 60 AsnGly Glu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Phe Ala Gly 65 70 75 80Ser Asn Lys Tyr Asp Gly His Ser Asn Lys Ser Gln Tyr Glu Ile Ile 85 90 95Thr Gln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile Gln 100 105110 85 1114 DNA Bacillus thuringiensis 85 atgttagata ctaataaagtttatgaaata agcaatcatg ctaatggact atatgcagca 60 acttatttaa gtttagatgattcaggtgtt agtttaatga ataaaaatga tgatgatatt 120 gatgattata acttaaaatggtttttattt cctattgatg atgatcaata tattattaca 180 agctatgcag caaataattgtaaagtttgg aatgttaata atgataaaat aaatgtttcg 240 acttattctt caacaaattcaatacaaaaa tggcaaataa aagctaatgg ttcttcatat 300 gtaatacaaa gtgataatggaaaagtctta acagcaggaa ccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatcctcaaataat cccaatcaac aatggaattt aacttctgta 420 caaacaattc aacttccacgaaaacctata atagatacaa aattaaaaga ttatcccaaa 480 tattcaccaa ctggaaatatagataatgga acatctcctc aattaatggg atggacatta 540 gtaccttgta ttatggtaaatgatccaaat atagataaaa atactcaaat taaaactact 600 ccatattata ttttaaaaaaatatcaatat tggcaacgag cagtaggaag taatgtagct 660 ttacgtccac atgaaaaaaaatcatatact tatgaatggg gcacagaaat agatcaaaaa 720 acaacaatta taaatacattaggatttcaa atcaatatag attcaggaat gaaatttgat 780 ataccagaag taggtggaggtacagatgaa ataaaaacac aactaaatga agaattaaaa 840 atagaatata gtcatgaaactaaaataatg gaaaaatatc aagaacaatc tgaaatagat 900 aatccaactg atcaatcaatgaattctata ggatttctta ctattacttc cttagaatta 960 tatagatata atggctcagaaattcgtata atgcaaattc aaacctcaga taatgatact 1020 tataatgtta cttcttatccaaatcatcaa caagctttat tacttcttac aaatcattca 1080 tatgaagaag ttgaagaaataacaagggcg aatt 1114 86 371 PRT Bacillus thuringiensis 86 Met Leu AspThr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu TyrAla Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met AsnLys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu PhePro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn AsnCys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 ThrTyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 GlySer Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130135 140 Leu Pro Arg Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro GlnLeu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp ProAsn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr IleLeu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn ValAla Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp GlyThr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu GlyPhe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro GluVal Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu GluLeu Lys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys TyrGln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met AsnSer Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr ArgTyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 AspAsn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360365 Arg Ala Asn 370 87 341 DNA Bacillus thuringiensis 87 atgtcagctggcgaagttca tattgaaata aacaataaaa cacgtcatac attacaatta 60 gaggataaaactaaacttag cggcggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaattaaaacatttgt agcagaatca catggtttta tgacaggagt agaaggtatt 180 atatattttagtgtaaacgg agacgcagaa attagtttac attttgacaa tccttatata 240 ggttctaataaatgtgatgg ttcttctgat aaacctgaat atgaagttat tactcaaagc 300 ggatcaggagataaatctca tgtcacttat acaattcaaa c 341 88 113 PRT Bacillus thuringiensis88 Met Ser Ala Gly Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 510 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 2025 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 3540 45 Glu Ser His Gly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 5055 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 6570 75 80 Gly Ser Asn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile100 105 110 Gln 89 1186 DNA Bacillus thuringiensis 89 atgttagatacaaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatca 60 acttatttaagtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120 gatgattacaatttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180 agctatggagctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240 acttattcttcaacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300 ataatacaaagtgataatgg aaaggtctta acagcaggag taggtcaatc tcttggaata 360 gtacgcctaactgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420 caaacaattcaactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480 tattcagaaaccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540 gtaccttgtattatggtaaa tgattcaaaa atagataaaa acactcaaat taaaactact 600 ccatattatatttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660 ttacttccacatcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720 acaactattattaatacagt aggattgcaa attaatatag attcaggaat gaaatttgaa 780 gtaccagaagtaggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840 gttgaatatagcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900 aatccaactaatcaaccaat gaattctata ggacttctta tttatacttc tttagaatta 960 tatcgatataacggrcagaa attaagataa tggacataga aacttcagat catgatactt 1020 acactcttacttcttatcca aatcataaag aagcattatt acttctcaca aaccattctt 1080 atgaagaagtagaagaaatt acaagggcga attccagcac actggcggcc gttactagtg 1140 gatccgagctcggtaccaag cttggcgtgt caggtcaaag ggttca 1186 90 392 PRT Bacillusthuringiensis 90 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu AlaAsn Gly 1 5 10 15 Leu Tyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser GlyVal Ser Leu 20 25 30 Met Ser Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn LeuLys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr SerTyr Gly Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Lys Asn Asp Lys IleAsn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp GlnIle Lys Ala Lys 85 90 95 Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly LysVal Leu Thr Ala 100 105 110 Gly Val Gly Gln Ser Leu Gly Ile Val Arg LeuThr Asp Glu Phe Pro 115 120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu ThrPro Val Gln Thr Ile Gln 130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp GluLys Leu Lys Asp His Pro Glu 145 150 155 160 Tyr Ser Glu Thr Gly Asn IleAsn Pro Lys Thr Thr Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val ProCys Ile Met Val Asn Asp Ser Lys Ile Asp 180 185 190 Lys Asn Thr Gln IleLys Thr Thr Pro Tyr Tyr Ile Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp AsnLeu Ala Lys Gly Ser Asn Val Ser Leu Leu Pro His 210 215 220 Gln Lys ArgSer Tyr Asp Tyr Glu Trp Gly Thr Glu Lys Asn Gln Lys 225 230 235 240 ThrThr Ile Ile Asn Thr Val Gly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255Met Lys Phe Glu Val Pro Glu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265270 Thr Gln Leu Thr Glu Glu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275280 285 Ile Met Thr Lys Tyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn290 295 300 Gln Pro Met Asn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu GluLeu 305 310 315 320 Tyr Arg Tyr Asn Gly Gln Lys Leu Arg Trp Thr Lys LeuGln Ile Met 325 330 335 Ile Leu Thr Leu Leu Leu Leu Ile Gln Ile Ile LysLys His Tyr Tyr 340 345 350 Phe Ser Gln Thr Ile Leu Met Lys Lys Lys LysLeu Gln Gly Arg Ile 355 360 365 Pro Ala His Trp Arg Pro Leu Leu Val AspPro Ser Ser Val Pro Ser 370 375 380 Leu Ala Cys Gln Val Lys Gly Phe 385390 91 341 DNA Bacillus thuringiensis 91 atgtcagcag ccgaagtacatattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgcacatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttcaagcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatggagaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaata aatattctggacgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaataatcatgat catgtaactt a 341 92 113 PRT Bacillus thuringiensis 92 Met SerAla Ala Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 ThrLeu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 IleThr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 GlySer Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 IleAsn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105110 Thr 93 341 DNA Bacillus thuringiensis 93 atgtcagatc gcgaagtacatattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgcacatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttcaagcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatggagaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaata aatattctggacgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaataatcatgat catgtaactt a 341 94 113 PRT Bacillus thuringiensis 94 Met SerAsp Arg Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 ThrLeu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 IleThr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 GlySer Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 IleAsn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105110 Thr 95 353 DNA Bacillus thuringiensis 95 atgtcagcac gtgaagtacatattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgcacatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttcaagcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatggagaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaata aatattctggacgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaataatcatgat catgtaacat atacgattca aac 353 96 117 PRT Bacillusthuringiensis 96 Met Ser Ala Arg Glu Val His Ile Glu Ile Ile Asn His ThrGly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His GlyGlu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp LeuPhe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile IleIle Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe Asp AsnPro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp AspAsp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn AsnHis Asp His Val 100 105 110 Thr Tyr Thr Ile Gln 115 97 353 DNA Bacillusthuringiensis 97 atgtcagctc gtgaagtaca tattgaaata ataaatcata caggtcataccttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattatta cacccgtgaatgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttt tgacaggagtagaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttac attttgacaatccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttataactgaagca 300 agagcagaac atagagctaa taatcatgat catgtgacat atacaattcaaac 353 98 117 PRT Bacillus thuringiensis 98 Met Ser Ala Arg Glu Val HisIle Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp LysArg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn ValPro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val LeuThr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile GluIle Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys TyrSer Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala ArgAla Glu His Arg Ala Asn Asn His Asp His Val 100 105 110 Thr Tyr Thr IleGln 115 99 353 DNA Bacillus thuringiensis 99 atgtcaggtc gcgaagttcatattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgcacatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttcaagcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatggagaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaata aatattctggacgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaataatcatgat catgtaacat atacgattca aac 353 100 117 PRT Bacillusthuringiensis 100 Met Ser Gly Arg Glu Val His Ile Glu Ile Ile Asn HisThr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala HisGly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser AspLeu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly IleIle Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe AspAsn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser AspAsp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu His Arg Ala AsnAsn His Asp His Val 100 105 110 Thr Tyr Thr Ile Gln 115 101 353 DNABacillus thuringiensis 101 atgtcagctc gtgaagtaca tattgaaata ataaatcatacaggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattattacacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttttgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaa attaccttacattttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgattataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgat catgttacgtatacaattca aac 353 102 117 PRT Bacillus thuringiensis 102 Met Ser AlaArg Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr LeuGln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile ThrPro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly SerAsp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile AsnGly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 GlySer Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 IleThr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105 110Thr Tyr Thr Ile Gln 115 103 353 DNA Bacillus thuringiensis 103atgtcaggtc gcgaagtaga tattgaaata ataaatcata caggtcatac cttacaaatg 60gataaaagaa ctagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120aattcttctg atttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180ataatttata ctataaatgg agaaatagaa attaccttac attttgacaa tccttatgca 240ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttat aactgaagcg 300agagcagaac atagagctaa taatcatgat catgtaacat atactattca gac 353 104 117PRT Bacillus thuringiensis 104 Met Ser Gly Arg Glu Val Asp Ile Glu IleIle Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr ArgLeu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn AsnSer Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly ValGlu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr LeuHis Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly ArgSer Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu HisArg Ala Asn Asn His Asp His Val 100 105 110 Thr Tyr Thr Ile Gln 115 105353 DNA Bacillus thuringiensis 105 atgtcagcac gtgaagtaca tattgaaataataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaatggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttctgatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaaattaccttac attttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagtgatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgatcatgtaacat ataccattca aac 353 106 117 PRT Bacillus thuringiensis 106 MetSer Ala Arg Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 7580 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 9095 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100105 110 Thr Tyr Thr Ile Gln 115 107 341 DNA Bacillus thuringiensis 107atgtcaggtc gcgaagttca tattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caagacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgacaa tccttattca 240ggttctaata aatatgatgg gcattccaat aaaaatcaat atgaagttat tacccaagga 300ggatcaggaa atcaatctca tctgacgtat acaattcaaa c 341 108 113 PRT Bacillusthuringiensis 108 Met Ser Gly Arg Glu Val His Ile Asp Val Asn Asn LysThr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Arg Leu Asp GlyGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly ThrIle Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe AspAsn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn LysAsn Gln Tyr Glu Val 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser HisLeu Thr Tyr Thr Ile 100 105 110 Gln 109 1114 DNA Bacillus thuringiensis109 atgttagata ctaataaagt atatgaaata agtaattatg ctaatggatt acatgcagca 60acttatttaa gtttagatga ttcaggtgtt agtttaatga ataaaaatga tgatgatatt 120gatgactata atttaaggtg gtttttattt cctattgatg ataatcaata tattattaca 180agctacgcag cgaataattg taaggtttgg aatgttaata atgataaaat aaatgtttca 240acttattctt caacaaactc gatacagaaa tggcaaataa aagctaatgc ttcttcgtat 300gtaatacaaa gtaataatgg gaaagttcta acagcaggaa ccggtcaatc tcttggatta 360atacgtttaa cggatgaatc accagataat cccaatcaac aatggaattt aactcctgta 420caaacaattc aactcccacc aaaacctaca atagatacaa agttaaaaga ttaccccaaa 480tattcacaaa ctggcaatat agacaaggga acacctcctc aattaatggg atggacatta 540ataccttgta ttatggtaaa tgatccaaat atagataaaa acactcaaat caaaactact 600ccatattata ttttaaaaaa atatcaatat tggcaacaag cagtaggaag taatgtagct 660ttacgtccgc atgaaaaaaa atcatatgct tatgagtggg gtacagaaat agatcaaaaa 720acaactatca ttaatacatt aggatttcag attaatatag attcgggaat ggaatttgat 780ataccagaag taggtggagg tacagatgaa ataaaaacac aattaaacga agaattaaaa 840atagaatata gccgtgaaac caaaataatg gaaaaatatc aggaacaatc agagatagat 900aatccaactg atcaatcaat gaattctata ggattcctca ctattacttc tttagaatta 960tatcgatata atggttcgga aattagtgta atgaaaattc aaacttcaga taatgatact 1020tacaatgtga cctcttatcc agatcatcaa caagctctat tacttcttac aaatcattca 1080tatgaacaag tacaagaaat aacaagggcg aatt 1114 110 371 PRT Bacillusthuringiensis 110 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn TyrAla Asn Gly 1 5 10 15 Leu His Ala Ala Thr Tyr Leu Ser Leu Asp Asp SerGly Val Ser Leu 20 25 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr AsnLeu Arg Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asp Asn Gln Tyr Ile Ile ThrSer Tyr Ala Ala 50 55 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp LysIle Asn Val Ser 65 70 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys TrpGln Ile Lys Ala Asn 85 90 95 Ala Ser Ser Tyr Val Ile Gln Ser Asn Asn GlyLys Val Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ser Leu Gly Leu Ile ArgLeu Thr Asp Glu Ser Pro 115 120 125 Asp Asn Pro Asn Gln Gln Trp Asn LeuThr Pro Val Gln Thr Ile Gln 130 135 140 Leu Pro Pro Lys Pro Thr Ile AspThr Lys Leu Lys Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Gln Thr Gly AsnIle Asp Lys Gly Thr Pro Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu IlePro Cys Ile Met Val Asn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr GlnIle Lys Thr Thr Pro Tyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr TrpGln Gln Ala Val Gly Ser Asn Val Ala Leu Arg Pro His 210 215 220 Glu LysLys Ser Tyr Ala Tyr Glu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240Thr Thr Ile Ile Asn Thr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250255 Met Glu Phe Asp Ile Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260265 270 Thr Gln Leu Asn Glu Glu Leu Lys Ile Glu Tyr Ser Arg Glu Thr Lys275 280 285 Ile Met Glu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro ThrAsp 290 295 300 Gln Ser Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser LeuGlu Leu 305 310 315 320 Tyr Arg Tyr Asn Gly Ser Glu Ile Ser Val Met LysIle Gln Thr Ser 325 330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr ProAsp His Gln Gln Ala 340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr GluGln Val Gln Glu Ile Thr 355 360 365 Arg Ala Asn 370 111 341 DNA Bacillusthuringiensis 111 atgtcagctc gtgaagtaca tattgaaata aacaataaaa cacgtcatacattacaatta 60 gaggataaaa ctaaacttag cggcggtaga tggcgaacat cacctacaaatgttgctcgt 120 gatacaatta aaacatttgt agcagaatca catggtttta tgacaggagtagaaggtatt 180 atatatttta gtgtaaacgg agacgcagaa attagtttac attttgacaatccttatata 240 ggttctaata aatgtgatgg ttcttctgat aaacctgaat atgaagttattactcaaagc 300 ggatcaggag ataaatctca tgttacatat acaattcaga c 341 112 113PRT Bacillus thuringiensis 112 Met Ser Ala Arg Glu Val His Ile Glu IleAsn Asn Lys Thr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr LysLeu Ser Gly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg AspThr Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly ValGlu Gly Ile Ile Tyr Phe Ser 50 55 60 Val Asn Gly Asp Ala Glu Ile Ser LeuHis Phe Asp Asn Pro Tyr Ile 65 70 75 80 Gly Ser Asn Lys Cys Asp Gly SerSer Asp Lys Pro Glu Tyr Glu Val 85 90 95 Ile Thr Gln Ser Gly Ser Gly AspLys Ser His Val Thr Tyr Thr Ile 100 105 110 Gln 113 360 DNA Bacillusthuringiensis 113 atgtcagctc gcgaagtaca cattgaaata aacaataaaa cacgtcatacattacaatta 60 gaggataaaa ctaaacttag cggcggtaga tggcgaacat cacctacaaatgttgctcgt 120 gatacaatta aaacatttgt agcagaatca catggtttta tgacaggagtagaaggtatt 180 atatatttta gtgtaaacgg agacgcagaa attagtttac attttgacaatccttatata 240 ggttctaata aatgtgatgg ttcttctgat aaacctgaat atgaagttattactcaaagc 300 ggatcaggag ataaatctca tgtgacatat actattcaga cagtatctttacgattataa 360 114 119 PRT Bacillus thuringiensis 114 Met Ser Ala ArgGlu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15 Thr Leu GlnLeu Glu Asp Lys Thr Lys Leu Ser Gly Gly Arg Trp Arg 20 25 30 Thr Ser ProThr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser HisGly Phe Met Thr Gly Val Glu Gly Ile Ile Tyr Phe Ser 50 55 60 Val Asn GlyAsp Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Ile 65 70 75 80 Gly SerAsn Lys Cys Asp Gly Ser Ser Asp Lys Pro Glu Tyr Glu Val 85 90 95 Ile ThrGln Ser Gly Ser Gly Asp Lys Ser His Val Thr Tyr Thr Ile 100 105 110 GlnThr Val Ser Leu Arg Leu 115 115 1158 DNA Bacillus thuringiensis 115atgttagata ctaataaagt ttatgaaata agcaatcttg ctaatggatt atatacatca 60acttatttaa gtcttgatga ttcaggtgtt agtttaatga gtaaaaagga tgaagatatt 120gatgattaca atttaaaatg gtttttattt cctattgata ataatcaata tattattaca 180agctatggag ctaataattg taaagtttgg aatgttaaaa atgataaaat aaatgtttca 240acttattctt caacaaactc tgtacaaaaa tggcaaataa aagctaaaga ttcttcatat 300ataatacaaa gtgataatgg aaaggtctta acagcaggag taggtgaatc tcttggaata 360gtacgcctaa ctgatgaatt tccagagaat tctaaccaac aatggaattt aactcctgta 420caaacaattc aactcccaca aaaacctaaa atagatgaaa aattaaaaga tcatcctgaa 480tattcagaaa ccggaaatat aaatcctaaa acaactcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgattcagga atagataaaa acactcaaat taaaactact 600ccatattata tttttaaaaa atataaatac tggaatctag caaaaggaag taatgtatct 660ttacttccac atcaaaaaag atcatatgat tatgaatggg gtacagaaaa aaatcaaaaa 720acatctatta ttaatacagt aggattgcaa attaatatag attcaggaat gaaatttgaa 780gtaccagaag taggaggagg tacagaagac ataaaaacac aattaactga agaattaaaa 840gttgaatata gcactgaaac caaaataatg acgaaatatc aagaacactc agagatagat 900aatccaacta atcaaccaat gaattctata ggacttctta tttatacttc tttagaatta 960tatcgatata acggtacaga aattaagata atggacatag aaacttcaga tcatgatact 1020tacactctta cttcttatcc aaatcataaa gaagcattat tacttctcac aaaccattcg 1080tatgaagaag tagaagaaat aacaaaaata cctaagcata cacttataaa attgaaaaaa 1140cattatttta aaaaataa 1158 116 385 PRT Bacillus thuringiensis 116 Met LeuAsp Thr Asn Lys Val Tyr Glu Ile Ser Asn Leu Ala Asn Gly 1 5 10 15 LeuTyr Thr Ser Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 MetSer Lys Lys Asp Glu Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 LeuPhe Pro Ile Asp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 AsnAsn Cys Lys Val Trp Asn Val Lys Asn Asp Lys Ile Asn Val Ser 65 70 75 80Thr Tyr Ser Ser Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95Asp Ser Ser Tyr Ile Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105110 Gly Val Gly Glu Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Phe Pro 115120 125 Glu Asn Ser Asn Gln Gln Trp Asn Leu Thr Pro Val Gln Thr Ile Gln130 135 140 Leu Pro Gln Lys Pro Lys Ile Asp Glu Lys Leu Lys Asp His ProGlu 145 150 155 160 Tyr Ser Glu Thr Gly Asn Ile Asn Pro Lys Thr Thr ProGln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn AspSer Gly Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr TyrIle Phe Lys Lys Tyr 195 200 205 Lys Tyr Trp Asn Leu Ala Lys Gly Ser AsnVal Ser Leu Leu Pro His 210 215 220 Gln Lys Arg Ser Tyr Asp Tyr Glu TrpGly Thr Glu Lys Asn Gln Lys 225 230 235 240 Thr Ser Ile Ile Asn Thr ValGly Leu Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Glu Val ProGlu Val Gly Gly Gly Thr Glu Asp Ile Lys 260 265 270 Thr Gln Leu Thr GluGlu Leu Lys Val Glu Tyr Ser Thr Glu Thr Lys 275 280 285 Ile Met Thr LysTyr Gln Glu His Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Pro MetAsn Ser Ile Gly Leu Leu Ile Tyr Thr Ser Leu Glu Leu 305 310 315 320 TyrArg Tyr Asn Gly Thr Glu Ile Lys Ile Met Asp Ile Glu Thr Ser 325 330 335Asp His Asp Thr Tyr Thr Leu Thr Ser Tyr Pro Asn His Lys Glu Ala 340 345350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355360 365 Lys Ile Pro Lys His Thr Leu Ile Lys Leu Lys Lys His Tyr Phe Lys370 375 380 Lys 385 117 341 DNA Bacillus thuringiensis 117 atgtcagcacgccaacttca tattgatgta aataataaga caggtcatac attacaatta 60 gaagataaaacaaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120 gatcaaattaaaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180 atatattatagtataaatgg agaagcagaa attagtttat attttgacaa tccttattca 240 ggttctaataaatatgatgg gcattctaat aaaaatcaat atgaagttat tacccaagga 300 ggatcaggaaatcaatctca tgtgacttat acgattcaca c 341 118 113 PRT Bacillusthuringiensis 118 Met Ser Ala Arg Gln Leu His Ile Asp Val Asn Asn LysThr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp GlyGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly ThrIle Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe AspAsn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn LysAsn Gln Tyr Glu Val 85 90 95 Ile Thr Gln Gly Gly Ser Gly Asn Gln Ser HisVal Thr Tyr Thr Ile 100 105 110 His 119 341 DNA Bacillus thuringiensis119 atgtcaggtc gtgaagttca tattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240ggttctaata aatatgatgg gcattccaat aaacctcaat atgaagttac tacccaagga 300ggatcaggaa atcaatctca tgtaacgtat actattcaaa c 341 120 113 PRT Bacillusthuringiensis 120 Met Ser Gly Arg Glu Val His Ile Asp Val Asn Asn LysThr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp GlyGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly ThrIle Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe AspAsn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn LysPro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly Asn Gln Ser HisVal Thr Tyr Thr Ile 100 105 110 Gln 121 341 DNA Bacillus thuringiensis121 atgtcaggtc gcgaagttga cattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240ggttctaata aatatgatgg gcattccaat aaacctcaat atgaagttac tacccaagga 300ggatcaggaa atcaatctca tgtcacatat acgattcaaa c 341 122 113 PRT Bacillusthuringiensis 122 Met Ser Gly Arg Glu Val Asp Ile Asp Val Asn Asn LysThr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp GlyGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly ThrIle Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe AspAsn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn LysPro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly Asn Gln Ser HisVal Thr Tyr Thr Ile 100 105 110 Gln 123 341 DNA Bacillus thuringiensis123 atgtcagcac gtgaagtaga tattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240ggttctaata aatatgatgg gcattccaat aaacctcaat atgaagttac tacccaagga 300ggatcaggaa atcaatctca tgtaacgtat acgattcaaa c 341 124 113 PRT Bacillusthuringiensis 124 Met Ser Ala Arg Glu Val Asp Ile Asp Val Asn Asn LysThr Gly His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp GlyGly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Asn Asp Gln Ile LysThr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Thr Glu Gly ThrIle Tyr Tyr Ser 50 55 60 Ile Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe AspAsn Pro Tyr Ser 65 70 75 80 Gly Ser Asn Lys Tyr Asp Gly His Ser Asn LysPro Gln Tyr Glu Val 85 90 95 Thr Thr Gln Gly Gly Ser Gly Asn Gln Ser HisVal Thr Tyr Thr Ile 100 105 110 Gln 125 1103 DNA Bacillus thuringiensis125 atgttagata ctaataaagt ttatgaaata agtaatcatg ctaatggact atatgcagca 60acttatttaa gtttagatga ttcaggtgtt agtttaatga ataaaaatga tgatgatatt 120gatgattata acttaaaatg gtttttattt cctattgatg atgatcaata tattattaca 180agctatgcag caaataattg taaagtttgg aatgttaata atgataaaat aaatgtttcg 240acttattctt caacaaattc aatacaaaaa tggcaaataa aagctaatgg ttcttcatat 300gtaatacaaa gtgataatgg aaaagtctta acagcaggaa ccggtcaagc tcttggattg 360atacgtttaa ctgatgaatc ctcaaataat cccaatcaac aatggaattt aacttctgta 420caaacaattc aacttccaca aaaacctata atagatacaa aattaaaaga ttatcccaaa 480tattcaccaa ctggaaatat agataatgga acatctcctc aattaatggg atggacatta 540gtaccttgta ttatggtaaa tgatccaaat atagataaaa atactcaaat taaaactact 600ccatattata ttttaaaaaa atatcaatat tggcaacgag cagtaggaag taatgtagct 660ttacgtccac atgaagaaaa atcatatact tatgaatggg gaacagaaat agatcaaaaa 720acaacaatca taaatacatt aggatttcaa atcaatatag attcaggaat gaaatttgat 780ataccagaag taggtggagg tacagatgaa ataaaaacac aactaaatga agaattaaaa 840atagaatata gtcgtgaaac taaaataatg gaaaaatatc aagaacaatc tgaaatagat 900aatccaactg atcaaccaat gaattctata ggatttctta ctattacttc tttagaatta 960tatagatata atggctcaga aattcgtata atgcaaattc aaacctcaga taatgatact 1020tataatgtta cttcttatcc agatcatcaa caagctttat tacttcttac aaatcattca 1080tatgaagaac ttgaagaaat tag 1103 126 367 PRT Bacillus thuringiensis 126Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 1015 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 2530 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 4045 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 5560 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 7075 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 8590 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu SerSer 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln ThrIle Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys AspTyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly ThrSer Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met ValAsn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr ProTyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val GlySer Asn Val Ala Leu Arg Pro His 210 215 220 Glu Glu Lys Ser Tyr Thr TyrGlu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile AsnThr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe AspIle Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln LeuAsn Glu Glu Leu Lys Ile Glu Tyr Ser Arg Glu Thr Lys 275 280 285 Ile MetGlu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 GlnPro Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asp His Gln Gln Ala340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Leu Glu Glu Ile355 360 365 127 369 DNA Artificial Sequence Description of ArtificialSequence gene sequence optimized for expression in Zea mays 127atgtccgccc gcgaggtgca catcgacgtg aacaacaaga ccggccacac cctccagctg 60gaggacaaga ccaagctcga cggcggcagg tggcgcacct ccccgaccaa cgtggccaac 120gaccagatca agaccttcgt ggccgaatcc aacggcttca tgaccggcac cgagggcacc 180atctactact ccatcaacgg cgaggccgag atcagcctct acttcgacaa cccgttcgcc 240ggctccaaca aatacgacgg ccactccaac aagtcccagt acgagatcat cacccagggc 300ggctccggca accagtccca cgtgacctac accatccaga ccacctcctc ccgctacggc 360cacaagtcc 369 128 1149 DNA Artificial Sequence Description of ArtificialSequence gene sequence optimized for expression in Zea mays 128atgctcgaca ccaacaaggt gtacgagatc agcaaccacg ccaacggcct ctacgccgcc 60acctacctct ccctcgacga ctccggcgtg tccctcatga acaagaacga cgacgacatc 120gacgactaca acctcaagtg gttcctcttc ccgatcgacg acgaccagta catcatcacc 180tcctacgccg ccaacaactg caaggtgtgg aacgtgaaca acgacaagat caacgtgtcc 240acctactcct ccaccaactc catccagaag tggcagatca aggccaacgg ctcctcctac 300gtgatccagt ccgacaacgg caaggtgctc accgccggca ccggccaggc cctcggcctc 360atccgcctca ccgacgagtc ctccaacaac ccgaaccagc aatggaacct gacgtccgtg 420cagaccatcc agctcccgca gaagccgatc atcgacacca agctcaagga ctacccgaag 480tactccccga ccggcaacat cgacaacggc acctccccgc agctcatggg ctggaccctc 540gtgccgtgca tcatggtgaa cgacccgaac atcgacaaga acacccagat caagaccacc 600ccgtactaca tcctcaagaa gtaccagtac tggcagaggg ccgtgggctc caacgtcgcg 660ctccgcccgc acgagaagaa gtcctacacc tacgagtggg gcaccgagat cgaccagaag 720accaccatca tcaacaccct cggcttccag atcaacatcg acagcggcat gaagttcgac 780atcccggagg tgggcggcgg taccgacgag atcaagaccc agctcaacga ggagctcaag 840atcgagtact cccacgagac gaagatcatg gagaagtacc aggagcagtc cgagatcgac 900aacccgaccg accagtccat gaactccatc ggcttcctca ccatcacctc cctggagctc 960taccgctaca acggctccga gatccgcatc atgcagatcc agacctccga caacgacacc 1020tacaacgtga cctcctaccc gaaccaccag caggccctgc tgctgctgac caaccactcc 1080tacgaggagg tggaggagat caccaacatc ccgaagtcca ccctcaagaa gctcaagaag 1140tactacttc 1149 129 357 DNA Artificial Sequence Description of ArtificialSequence maize-optimized gene sequence 129 atgtccgccc gcgaggtgcacatcgagatc aacaacaaga cccgccacac cctccagctc 60 gaggacaaga ccaagctctccggcggcagg tggcgcacct ccccgaccaa cgtggcccgc 120 gacaccatca agacgttcgtggcggagtcc cacggcttca tgaccggcgt cgagggcatc 180 atctacttct ccgtgaacggcgacgccgag atctccctcc acttcgacaa cccgtacatc 240 ggctccaaca agtccgacggctcctccgac aagcccgagt acgaggtgat cacccagtcc 300 ggctccggcg acaagtcccacgtgacctac accatccaga ccgtgtccct ccgcctc 357 130 119 PRT ArtificialSequence Description of Artificial Sequence protein encoded bymaize-optimized gene 130 Met Ser Ala Arg Glu Val His Ile Glu Ile Asn AsnLys Thr Arg His 1 5 10 15 Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu SerGly Gly Arg Trp Arg 20 25 30 Thr Ser Pro Thr Asn Val Ala Arg Asp Thr IleLys Thr Phe Val Ala 35 40 45 Glu Ser His Gly Phe Met Thr Gly Val Glu GlyIle Ile Tyr Phe Ser 50 55 60 Val Asn Gly Asp Ala Glu Ile Ser Leu His PheAsp Asn Pro Tyr Ile 65 70 75 80 Gly Ser Asn Lys Ser Asp Gly Ser Ser AspLys Pro Glu Tyr Glu Val 85 90 95 Ile Thr Gln Ser Gly Ser Gly Asp Lys SerHis Val Thr Tyr Thr Ile 100 105 110 Gln Thr Val Ser Leu Arg Leu 115 13121 DNA Artificial Sequence Description of Artificial Sequenceoligonucleotide primer 131 atgtcagctc gcgaagtaca c 21 132 22 DNAArtificial Sequence Description of Artificial Sequence oligonucleotideprimer 132 gtccatccca ttaattgagg ag 22 133 399 DNA Bacillusthuringiensis 133 atgtcagcac gtgaagtaca cattgaaata ataaatcata caggtcataccttacaaatg 60 gataaaagaa ctagacttgc acatggtgaa tggattatta cacccgtgaatgttccaaat 120 aattcttctg atttatttca agcaggttct gatggagttt tgacaggagtagaaggaata 180 ataatttata ctataaatgg agaaatagaa attcccttac attttgacaatccttatgca 240 ggttctaata aatattctgg acgttctagt gatgatgatt ataaagttataactgaagca 300 agagcagaac atagagctaa taatcatgat catgtaacat atacagttcaaagaaacata 360 tcacgatata ccaataaatt atgttctaat aactcctaa 399 134 132PRT Bacillus thuringiensis 134 Met Ser Ala Arg Glu Val His Ile Glu IleIle Asn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr ArgLeu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn AsnSer Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly ValGlu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Pro LeuHis Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly ArgSer Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu HisArg Ala Asn Asn His Asp His Val 100 105 110 Thr Tyr Thr Val Gln Arg AsnIle Ser Arg Tyr Thr Asn Lys Leu Cys 115 120 125 Ser Asn Asn Ser 130 1351164 DNA Bacillus thuringiensis 135 atgatagaaa ctaataagat atatgaaataagcaataaag ctaatggatt atatgcaact 60 acttatttaa gttttgataa ttcaggtgttagtttattaa ataaaaatga atctgatatt 120 aatgattata atttgaaatg gtttttatttcctattgata ataatcagta tattattaca 180 agttatggag taaataaaaa taaggtttggactgctaatg gtaataaaat aaatgttaca 240 acatattccg cagaaaattc agcacaacaatggcaaataa gaaacagttc ttctggatat 300 ataatagaaa ataataatgg gaaaattttaacggcaggaa caggccaatc attaggttta 360 ttatatttaa ctgatgaaat acctgaagattctaatcaac aatggaattt aacttcaata 420 caaacaattt cacttccttc acaaccaataattgatacaa cattagtaga ttaccctaaa 480 tattcaacga ccggtagtat aaattataatggtacagcac ttcaattaat gggatggaca 540 ctcataccat gtattatggt atacgataaaacgatagctt ctacacacac tcaaattaca 600 acaacccctt attatatttt gaaaaaatatcaacgttggg tacttgcaac aggaagtggt 660 ctatctgtac ctgcacatgt caaatcaactttcgaatacg aatggggaac agacacagat 720 caaaaaacca gtgtaataaa tacattaggttttcaaatta atacagatac aaaattaaaa 780 gctactgtac cagaagtagg tggaggtacaacagatataa gaacacaaat cactgaagaa 840 cttaaagtag aatatagtag tgaaaataaagaaatgcgaa aatataaaca aagctttgac 900 gtagacaact taaattatga tgaagcactaaatgctgtag gatttattgt tgaaacttca 960 ttcgaattat atcgaatgaa tggaaatgtccttataacaa gtataaaaac tacaaataaa 1020 gacacctata atacagttac ttatccaaatcataaagaag ttttattact tcttacaaat 1080 cattcttatg aagaagtaac agcactaactggcatttcca aagaaagact tcaaaatctt 1140 aaaaacaatt ggaaaaaaag ataa 1164136 387 PRT Bacillus thuringiensis 136 Met Ile Glu Thr Asn Lys Ile TyrGlu Ile Ser Asn Lys Ala Asn Gly 1 5 10 15 Leu Tyr Ala Thr Thr Tyr LeuSer Phe Asp Asn Ser Gly Val Ser Leu 20 25 30 Leu Asn Lys Asn Glu Ser AspIle Asn Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn AsnGln Tyr Ile Ile Thr Ser Tyr Gly Val 50 55 60 Asn Lys Asn Lys Val Trp ThrAla Asn Gly Asn Lys Ile Asn Val Thr 65 70 75 80 Thr Tyr Ser Ala Glu AsnSer Ala Gln Gln Trp Gln Ile Arg Asn Ser 85 90 95 Ser Ser Gly Tyr Ile IleGlu Asn Asn Asn Gly Lys Ile Leu Thr Ala 100 105 110 Gly Thr Gly Gln SerLeu Gly Leu Leu Tyr Leu Thr Asp Glu Ile Pro 115 120 125 Glu Asp Ser AsnGln Gln Trp Asn Leu Thr Ser Ile Gln Thr Ile Ser 130 135 140 Leu Pro SerGln Pro Ile Ile Asp Thr Thr Leu Val Asp Tyr Pro Lys 145 150 155 160 TyrSer Thr Thr Gly Ser Ile Asn Tyr Asn Gly Thr Ala Leu Gln Leu 165 170 175Met Gly Trp Thr Leu Ile Pro Cys Ile Met Val Tyr Asp Lys Thr Ile 180 185190 Ala Ser Thr His Thr Gln Ile Thr Thr Thr Pro Tyr Tyr Ile Leu Lys 195200 205 Lys Tyr Gln Arg Trp Val Leu Ala Thr Gly Ser Gly Leu Ser Val Pro210 215 220 Ala His Val Lys Ser Thr Phe Glu Tyr Glu Trp Gly Thr Asp ThrAsp 225 230 235 240 Gln Lys Thr Ser Val Ile Asn Thr Leu Gly Phe Gln IleAsn Thr Asp 245 250 255 Thr Lys Leu Lys Ala Thr Val Pro Glu Val Gly GlyGly Thr Thr Asp 260 265 270 Ile Arg Thr Gln Ile Thr Glu Glu Leu Lys ValGlu Tyr Ser Ser Glu 275 280 285 Asn Lys Glu Met Arg Lys Tyr Lys Gln SerPhe Asp Val Asp Asn Leu 290 295 300 Asn Tyr Asp Glu Ala Leu Asn Ala ValGly Phe Ile Val Glu Thr Ser 305 310 315 320 Phe Glu Leu Tyr Arg Met AsnGly Asn Val Leu Ile Thr Ser Ile Lys 325 330 335 Thr Thr Asn Lys Asp ThrTyr Asn Thr Val Thr Tyr Pro Asn His Lys 340 345 350 Glu Val Leu Leu LeuLeu Thr Asn His Ser Tyr Glu Glu Val Thr Ala 355 360 365 Leu Thr Gly IleSer Lys Glu Arg Leu Gln Asn Leu Lys Asn Asn Trp 370 375 380 Lys Lys Arg385 137 341 DNA Bacillus thuringiensis 137 atgtcagcag gtgaagttcatattgaaata aataataaaa cacgtcatac attacaatta 60 gaggataaaa ctaaacttaccagtggtaga tggcgaacat cacctacaaa tgttgctcgt 120 gatacaatta aaacatttgtagcagaatca catggtttta tgacaggaat agaaggtatt 180 atatatttta gcgtaaacggagaagcagaa attagtttac attttgacaa tccttatgta 240 ggttctaata aatatgatggttcttctgat aaagctgcat acgaagttat tgctcaaggt 300 ggatcagggg atatatctcatctaacatat acaattcaaa c 341 138 113 PRT Bacillus thuringiensis 138 MetSer Ala Gly Glu Val His Ile Glu Ile Asn Asn Lys Thr Arg His 1 5 10 15Thr Leu Gln Leu Glu Asp Lys Thr Lys Leu Thr Ser Gly Arg Trp Arg 20 25 30Thr Ser Pro Thr Asn Val Ala Arg Asp Thr Ile Lys Thr Phe Val Ala 35 40 45Glu Ser His Gly Phe Met Thr Gly Ile Glu Gly Ile Ile Tyr Phe Ser 50 55 60Val Asn Gly Glu Ala Glu Ile Ser Leu His Phe Asp Asn Pro Tyr Val 65 70 7580 Gly Ser Asn Lys Tyr Asp Gly Ser Ser Asp Lys Ala Ala Tyr Glu Val 85 9095 Ile Ala Gln Gly Gly Ser Gly Asp Ile Ser His Leu Thr Tyr Thr Ile 100105 110 Gln 139 1158 DNA Bacillus thuringiensis 139 atgttagatactaataaaat ttatgaaata agcaatcatg ctaatggatt atatacatca 60 acttatttaagtctggatga ttcaggtgtt agtttaatgg gtcaaaatga tgaggatata 120 gatgaatacaatttaaagtg gttcttattt ccaatagata ataatcaata tattattaca 180 agctatggagcgaataattg taaagtttgg aatgttaaaa atgataaagt aaatgtttca 240 acgtattctccaacaaactc agtacaaaaa tggcaaataa aagctaaaaa ttcttcatat 300 ataatacaaagtgagaatgg aaaagtctta acagcaggaa taggtcaatc tcttggaata 360 gtacgcttaaccgatgaatc atcagagagt tctaaccaac aatggaattt aatccctgta 420 caaacaatttcactcccaca aaaacctaaa atagataaaa aattaaaaga tcatcctgaa 480 tattcagaaaccggaaatat agctactgga acaattcctc aattaatggg atggacatta 540 gtaccttgtattatggtaaa tgatccaaaa ataggtaaaa acactcaaat taaaactact 600 ccatattatatttttaaaaa atatcaatac tggaaacgag caataggaag taatgtatct 660 ttacttccacatcaaaaaaa atcatatgat tatgagtggg gtacagaaga aaatcaaaaa 720 acaactattattaatacagt aggatttcaa attaatgtag attcaggaat gaagtttgag 780 gtaccagaagtaggaggagg tacagaagaa ataaaaacac aattaaatga agaattaaaa 840 gttgaatatagcactgacac caaaataatg aaaaaatatc aagaacactc agagatagat 900 aatccaactaatcaaacaac gaattctata ggatttctta cttttacttc tttagaatta 960 tatcgatataacggttcgga aattcgtata atgagaatgg aaacttcaga taatgatact 1020 tatactctgacctcttatcc aaatcataga gaagcattat tacttctcac aaatcattct 1080 tatcaagaagtaagccgaat tccagcacac tggcggccgt tactagtgga tccgagctcg 1140 gtaccaagcttggcgtaa 1158 140 385 PRT Bacillus thuringiensis 140 Met Leu Asp Thr AsnLys Ile Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr Thr SerThr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Gly Gln AsnAsp Glu Asp Ile Asp Glu Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe Pro IleAsp Asn Asn Gln Tyr Ile Ile Thr Ser Tyr Gly Ala 50 55 60 Asn Asn Cys LysVal Trp Asn Val Lys Asn Asp Lys Val Asn Val Ser 65 70 75 80 Thr Tyr SerPro Thr Asn Ser Val Gln Lys Trp Gln Ile Lys Ala Lys 85 90 95 Asn Ser SerTyr Ile Ile Gln Ser Glu Asn Gly Lys Val Leu Thr Ala 100 105 110 Gly IleGly Gln Ser Leu Gly Ile Val Arg Leu Thr Asp Glu Ser Ser 115 120 125 GluSer Ser Asn Gln Gln Trp Asn Leu Ile Pro Val Gln Thr Ile Ser 130 135 140Leu Pro Gln Lys Pro Lys Ile Asp Lys Lys Leu Lys Asp His Pro Glu 145 150155 160 Tyr Ser Glu Thr Gly Asn Ile Ala Thr Gly Thr Ile Pro Gln Leu Met165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro Lys IleGly 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile Phe LysLys Tyr 195 200 205 Gln Tyr Trp Lys Arg Ala Ile Gly Ser Asn Val Ser LeuLeu Pro His 210 215 220 Gln Lys Lys Ser Tyr Asp Tyr Glu Trp Gly Thr GluGlu Asn Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Val Gly Phe GlnIle Asn Val Asp Ser Gly 245 250 255 Met Lys Phe Glu Val Pro Glu Val GlyGly Gly Thr Glu Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu Leu LysVal Glu Tyr Ser Thr Asp Thr Lys 275 280 285 Ile Met Lys Lys Tyr Gln GluHis Ser Glu Ile Asp Asn Pro Thr Asn 290 295 300 Gln Thr Thr Asn Ser IleGly Phe Leu Thr Phe Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg Tyr AsnGly Ser Glu Ile Arg Ile Met Arg Met Glu Thr Ser 325 330 335 Asp Asn AspThr Tyr Thr Leu Thr Ser Tyr Pro Asn His Arg Glu Ala 340 345 350 Leu LeuLeu Leu Thr Asn His Ser Tyr Gln Glu Val Ser Arg Ile Pro 355 360 365 AlaHis Trp Arg Pro Leu Leu Val Asp Pro Ser Ser Val Pro Ser Leu 370 375 380Ala 385 141 399 DNA Bacillus thuringiensis 141 atgtcagatc gcgaagtacatattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgcacatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttcaagcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatggagaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaata aatattctggacgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaataatcatgat catgtaacat atacagttca aagaaacata 360 tcacgatata ccaataaattatgttctaat aactcctaa 399 142 132 PRT Bacillus thuringiensis 142 Met SerAsp Arg Glu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 ThrLeu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 IleThr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 GlySer Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 IleAsn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105110 Thr Tyr Thr Val Gln Arg Asn Ile Ser Arg Tyr Thr Asn Lys Leu Cys 115120 125 Ser Asn Asn Ser 130 143 871 DNA Bacillus thuringiensis 143atgatagaaa ctaataagat atatgaaata agcaataaag ctaatggatt atatgcaact 60acttatttaa gttttgataa ttcaggtgtt agtttattaa ataaaaatga atctgatatt 120aatgattata atttgaaatg gtttttattt cctattgata ataatcagta tattattaca 180agttatggag taaataaaaa taaggtttgg actgctaatg gtaataaaat aaatgttaca 240acatattccg cagaaaattc agcacaacaa tggcaaataa gaaacagttc ttctggatat 300ataatagaaa ataataatgg gaaaatttta acggcaggaa caggccaatc attaggttta 360ttatatttaa ctgatgaaat acctgaagat tctaatcaac aatggaattt aacttcaata 420caaacaattt cacttccttc acaaccaata attgatacaa cattagtaga ttaccctaaa 480tattcaacga ccggtagtat aaattataat ggtacagcac ttcaattaat gggatggaca 540ctcataccat gtattatggt atacgataaa acgatagctt ctacacacac tcaaattaca 600acaacccctt attatatttt gaaaaaatat caacgttggg tacttgcaac aggaagtggt 660ctatctgtac ctgcacatgt caaatcaact ttcgaatacg aatggggaac agacacagat 720caaaaaacca gtgtaataaa tacattaggt tttcaaatta atacagatac aaaattaaaa 780gctactgtac cagaagtagg tggaggtaca acagatataa gaacacaaat cactgaagaa 840cttaaagtag aatatagtag tgaaaataaa g 871 144 290 PRT Bacillusthuringiensis 144 Met Ile Glu Thr Asn Lys Ile Tyr Glu Ile Ser Asn LysAla Asn Gly 1 5 10 15 Leu Tyr Ala Thr Thr Tyr Leu Ser Phe Asp Asn SerGly Val Ser Leu 20 25 30 Leu Asn Lys Asn Glu Ser Asp Ile Asn Asp Tyr AsnLeu Lys Trp Phe 35 40 45 Leu Phe Pro Ile Asp Asn Asn Gln Tyr Ile Ile ThrSer Tyr Gly Val 50 55 60 Asn Lys Asn Lys Val Trp Thr Ala Asn Gly Asn LysIle Asn Val Thr 65 70 75 80 Thr Tyr Ser Ala Glu Asn Ser Ala Gln Gln TrpGln Ile Arg Asn Ser 85 90 95 Ser Ser Gly Tyr Ile Ile Glu Asn Asn Asn GlyLys Ile Leu Thr Ala 100 105 110 Gly Thr Gly Gln Ser Leu Gly Leu Leu TyrLeu Thr Asp Glu Ile Pro 115 120 125 Glu Asp Ser Asn Gln Gln Trp Asn LeuThr Ser Ile Gln Thr Ile Ser 130 135 140 Leu Pro Ser Gln Pro Ile Ile AspThr Thr Leu Val Asp Tyr Pro Lys 145 150 155 160 Tyr Ser Thr Thr Gly SerIle Asn Tyr Asn Gly Thr Ala Leu Gln Leu 165 170 175 Met Gly Trp Thr LeuIle Pro Cys Ile Met Val Tyr Asp Lys Thr Ile 180 185 190 Ala Ser Thr HisThr Gln Ile Thr Thr Thr Pro Tyr Tyr Ile Leu Lys 195 200 205 Lys Tyr GlnArg Trp Val Leu Ala Thr Gly Ser Gly Leu Ser Val Pro 210 215 220 Ala HisVal Lys Ser Thr Phe Glu Tyr Glu Trp Gly Thr Asp Thr Asp 225 230 235 240Gln Lys Thr Ser Val Ile Asn Thr Leu Gly Phe Gln Ile Asn Thr Asp 245 250255 Thr Lys Leu Lys Ala Thr Val Pro Glu Val Gly Gly Gly Thr Thr Asp 260265 270 Ile Arg Thr Gln Ile Thr Glu Glu Leu Lys Val Glu Tyr Ser Ser Glu275 280 285 Asn Lys 290 145 372 DNA Bacillus thuringiensis 145atgtcagcac gtgaagtaca cattgatgta aataataaga caggtcatac attacaatta 60gaagataaaa caaaacttga tggtggtaga tggcgaacat cacctacaaa tgttgctaat 120gatcaaatta aaacatttgt agcagaatca catggtttta tgacaggtac agaaggtact 180atatattata gtataaatgg agaagcagaa attagtttat attttgataa tccttattca 240ggttctaata aatatgatgg gcattccaat aaacctcaat atgaagttac tacccaagga 300ggatcaggaa atcaatctca tgttacgtat actattcaaa ctgcatcttc acgatatggg 360aataactcat aa 372 146 123 PRT Bacillus thuringiensis 146 Met Ser Ala ArgGlu Val His Ile Asp Val Asn Asn Lys Thr Gly His 1 5 10 15 Thr Leu GlnLeu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp Arg 20 25 30 Thr Ser ProThr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe Val Ala 35 40 45 Glu Ser HisGly Phe Met Thr Gly Thr Glu Gly Thr Ile Tyr Tyr Ser 50 55 60 Ile Asn GlyGlu Ala Glu Ile Ser Leu Tyr Phe Asp Asn Pro Tyr Ser 65 70 75 80 Gly SerAsn Lys Tyr Asp Gly His Ser Asn Lys Pro Gln Tyr Glu Val 85 90 95 Thr ThrGln Gly Gly Ser Gly Asn Gln Ser His Val Thr Tyr Thr Ile 100 105 110 GlnThr Ala Ser Ser Arg Tyr Gly Asn Asn Ser 115 120 147 1152 DNA Bacillusthuringiensis 147 atgttagata ctaataaagt ttatgaaata agtaatcatg ctaatggactatatgcagca 60 acttatttaa gtttagatga ttcaggtgtt agtttaatga ataaaaatgatgatgatatt 120 gatgattata acttaaaatg gtttttattt cctattgatg atgatcaatatattattaca 180 agctatgcag caaataattg taaagtttgg aatgttaata atgataaaataaatgtttcg 240 acttattctt caacaaattc aatacaaaaa tggcaaataa aagctaatggttcttcatat 300 gtaatacaaa gtgataatgg aaaagtctta acagcaggaa ccggtcaagctcttggattg 360 atacgtttaa ctgatgaatc ctcaaataat cccaatcaac aatggaatttaacttctgta 420 caaacaattc aacttccaca aaaacctata atagatacaa aattaaaagattatcccaaa 480 tattcaccaa ctggaaatat agataatgga acatctcctc aattaatgggatggacatta 540 gtaccttgta ttatggtaaa tgatccaaat atagataaaa atactcaaattaaaactact 600 ccatattata ttttaaaaaa atatcaatat tggcaacgag cagtaggaagtaatgtagct 660 ttacgtccac atgaaaaaaa atcatatact tatgaatggg gaacagaaatagatcaaaaa 720 acaacaatca taaatacatt aggatttcaa atcaatatag attcaggaatgaaatttgat 780 ataccagaag taggtggagg tacagatgaa ataaaaacac aactaaatgaagaattaaaa 840 atagaatata gtcgtgaaac taaaataatg gaaaaatatc aagaacaatctgaaatagat 900 aatccaactg atcaaccaat gaattctata ggatttctta ctattacttctttagaatta 960 tatagatata atggctcaga aattcgtata atgcaaattc aaacctcagataatgatact 1020 tataatgtta cttcttatcc agatcatcaa caagctttat tacttcttacaaatcattca 1080 tatgaagaag tagaagaaat aacaaatatt cctaaaagta cactaaaaaaattaaaaaaa 1140 tattattttt aa 1152 148 383 PRT Bacillus thuringiensis148 Met Leu Asp Thr Asn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 510 15 Leu Tyr Ala Ala Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 2025 30 Met Asn Lys Asn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 3540 45 Leu Phe Pro Ile Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 5055 60 Asn Asn Cys Lys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 6570 75 80 Thr Tyr Ser Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn85 90 95 Gly Ser Ser Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala100 105 110 Gly Thr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu SerSer 115 120 125 Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln ThrIle Gln 130 135 140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys AspTyr Pro Lys 145 150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly ThrSer Pro Gln Leu Met 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met ValAsn Asp Pro Asn Ile Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr ProTyr Tyr Ile Leu Lys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val GlySer Asn Val Ala Leu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr TyrGlu Trp Gly Thr Glu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile AsnThr Leu Gly Phe Gln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe AspIle Pro Glu Val Gly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln LeuAsn Glu Glu Leu Lys Ile Glu Tyr Ser Arg Glu Thr Lys 275 280 285 Ile MetGlu Lys Tyr Gln Glu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 GlnPro Met Asn Ser Ile Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315320 Tyr Arg Tyr Asn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325330 335 Asp Asn Asp Thr Tyr Asn Val Thr Ser Tyr Pro Asp His Gln Gln Ala340 345 350 Leu Leu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu IleThr 355 360 365 Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr TyrPhe 370 375 380 149 354 DNA Bacillus thuringiensis 149 atgtcagctcgcgaagttca tattgaaata ataaatcata caggtcatac cttacaaatg 60 gataaaagaactagacttgc acatggtgaa tggattatta cacccgtgaa tgttccaaat 120 aattcttctgatttatttca agcaggttct gatggagttt tgacaggagt agaaggaata 180 ataatttatactataaatgg agaaatagaa attaccttac attttgacaa tccttatgca 240 ggttctaataaatattctgg acgttctagt gatgatgatt ataaagttat aactgaagca 300 agagcagaacatagagctaa taatcatgat catgtgacat atacaattca aaca 354 150 113 PRTBacillus thuringiensis 150 Met Ser Ala Arg Glu Val His Ile Glu Ile IleAsn His Thr Gly His 1 5 10 15 Thr Leu Gln Met Asp Lys Arg Thr Arg LeuAla His Gly Glu Trp Ile 20 25 30 Ile Thr Pro Val Asn Val Pro Asn Asn SerSer Asp Leu Phe Gln Ala 35 40 45 Gly Ser Asp Gly Val Leu Thr Gly Val GluGly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn Gly Glu Ile Glu Ile Thr Leu HisPhe Asp Asn Pro Tyr Ala 65 70 75 80 Gly Ser Asn Lys Tyr Ser Gly Arg SerSer Asp Asp Asp Tyr Lys Val 85 90 95 Ile Thr Glu Ala Arg Ala Glu His ArgAla Asn Asn His Asp His Val 100 105 110 Thr 151 353 DNA Bacillusthuringiensis 151 152 113 PRT Bacillus thuringiensis 152 Met Ser Ala ArgGlu Val His Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15 Thr Leu GlnMet Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30 Ile Thr ProVal Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45 Gly Ser AspGly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60 Ile Asn GlyGlu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 75 80 Gly SerAsn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 90 95 Ile ThrGlu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100 105 110 Thr153 353 DNA Bacillus thuringiensis 153 atgtcagcac gcgaagtaga tattgaaataataaatcata caggtcatac cttacaaatg 60 gataaaagaa ctagacttgc acatggtgaatggattatta cacccgtgaa tgttccaaat 120 aattcttctg atttatttca agcaggttctgatggagttt tgacaggagt agaaggaata 180 ataatttata ctataaatgg agaaatagaaattaccttac attttgacaa tccttatgca 240 ggttctaata aatattctgg acgttctagtgatgatgatt ataaagttat aactgaagca 300 agagcagaac atagagctaa taatcatgatcatgtgactt atacaattca aac 353 154 113 PRT Bacillus thuringiensis 154 MetSer Ala Arg Glu Val Asp Ile Glu Ile Ile Asn His Thr Gly His 1 5 10 15Thr Leu Gln Met Asp Lys Arg Thr Arg Leu Ala His Gly Glu Trp Ile 20 25 30Ile Thr Pro Val Asn Val Pro Asn Asn Ser Ser Asp Leu Phe Gln Ala 35 40 45Gly Ser Asp Gly Val Leu Thr Gly Val Glu Gly Ile Ile Ile Tyr Thr 50 55 60Ile Asn Gly Glu Ile Glu Ile Thr Leu His Phe Asp Asn Pro Tyr Ala 65 70 7580 Gly Ser Asn Lys Tyr Ser Gly Arg Ser Ser Asp Asp Asp Tyr Lys Val 85 9095 Ile Thr Glu Ala Arg Ala Glu His Arg Ala Asn Asn His Asp His Val 100105 110 Thr 155 37 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 155 aaatattatt ttatgtcagc acgtgaagtacacattg 37 156 40 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 156 tctctggtac cttattatga tttatgcccatatcgtgagg 40 157 45 DNA Artificial Sequence Description of ArtificialSequence oligonucleotide primer 157 agagaactag taaaaaggag ataaccatgttagatactaa taaag 45 158 46 DNA Artificial Sequence Description ofArtificial Sequence oligonucleotide primer 158 cgtgctgaca taaaataatatttttttaat ttttttagtg tacttt 46 159 506 PRT Artificial SequenceDescription of Artificial Sequence fusion protein 159 Met Leu Asp ThrAsn Lys Val Tyr Glu Ile Ser Asn His Ala Asn Gly 1 5 10 15 Leu Tyr AlaAla Thr Tyr Leu Ser Leu Asp Asp Ser Gly Val Ser Leu 20 25 30 Met Asn LysAsn Asp Asp Asp Ile Asp Asp Tyr Asn Leu Lys Trp Phe 35 40 45 Leu Phe ProIle Asp Asp Asp Gln Tyr Ile Ile Thr Ser Tyr Ala Ala 50 55 60 Asn Asn CysLys Val Trp Asn Val Asn Asn Asp Lys Ile Asn Val Ser 65 70 75 80 Thr TyrSer Ser Thr Asn Ser Ile Gln Lys Trp Gln Ile Lys Ala Asn 85 90 95 Gly SerSer Tyr Val Ile Gln Ser Asp Asn Gly Lys Val Leu Thr Ala 100 105 110 GlyThr Gly Gln Ala Leu Gly Leu Ile Arg Leu Thr Asp Glu Ser Ser 115 120 125Asn Asn Pro Asn Gln Gln Trp Asn Leu Thr Ser Val Gln Thr Ile Gln 130 135140 Leu Pro Gln Lys Pro Ile Ile Asp Thr Lys Leu Lys Asp Tyr Pro Lys 145150 155 160 Tyr Ser Pro Thr Gly Asn Ile Asp Asn Gly Thr Ser Pro Gln LeuMet 165 170 175 Gly Trp Thr Leu Val Pro Cys Ile Met Val Asn Asp Pro AsnIle Asp 180 185 190 Lys Asn Thr Gln Ile Lys Thr Thr Pro Tyr Tyr Ile LeuLys Lys Tyr 195 200 205 Gln Tyr Trp Gln Arg Ala Val Gly Ser Asn Val AlaLeu Arg Pro His 210 215 220 Glu Lys Lys Ser Tyr Thr Tyr Glu Trp Gly ThrGlu Ile Asp Gln Lys 225 230 235 240 Thr Thr Ile Ile Asn Thr Leu Gly PheGln Ile Asn Ile Asp Ser Gly 245 250 255 Met Lys Phe Asp Ile Pro Glu ValGly Gly Gly Thr Asp Glu Ile Lys 260 265 270 Thr Gln Leu Asn Glu Glu LeuLys Ile Glu Tyr Ser His Glu Thr Lys 275 280 285 Ile Met Glu Lys Tyr GlnGlu Gln Ser Glu Ile Asp Asn Pro Thr Asp 290 295 300 Gln Ser Met Asn SerIle Gly Phe Leu Thr Ile Thr Ser Leu Glu Leu 305 310 315 320 Tyr Arg TyrAsn Gly Ser Glu Ile Arg Ile Met Gln Ile Gln Thr Ser 325 330 335 Asp AsnAsp Thr Tyr Asn Val Thr Ser Tyr Pro Asn His Gln Gln Ala 340 345 350 LeuLeu Leu Leu Thr Asn His Ser Tyr Glu Glu Val Glu Glu Ile Thr 355 360 365Asn Ile Pro Lys Ser Thr Leu Lys Lys Leu Lys Lys Tyr Tyr Phe Met 370 375380 Ser Ala Arg Glu Val His Ile Asp Val Asn Asn Lys Thr Gly His Thr 385390 395 400 Leu Gln Leu Glu Asp Lys Thr Lys Leu Asp Gly Gly Arg Trp ArgThr 405 410 415 Ser Pro Thr Asn Val Ala Asn Asp Gln Ile Lys Thr Phe ValAla Glu 420 425 430 Ser Asn Gly Phe Met Thr Gly Thr Glu Gly Thr Ile TyrTyr Ser Ile 435 440 445 Asn Gly Glu Ala Glu Ile Ser Leu Tyr Phe Asp AsnPro Phe Ala Gly 450 455 460 Ser Asn Lys Tyr Asp Gly His Ser Asn Lys SerGln Tyr Glu Ile Ile 465 470 475 480 Thr Gln Gly Gly Ser Gly Asn Gln SerHis Val Thr Tyr Thr Ile Gln 485 490 495 Thr Thr Ser Ser Arg Tyr Gly HisLys Ser 500 505 160 1521 DNA Artificial Sequence Description ofArtificial Sequence fusion gene 160 atgttagata ctaataaagt ttatgaaataagcaatcatg ctaatggact atatgcagca 60 acttatttaa gtttagatga ttcaggtgttagtttaatga ataaaaatga tgatgatatt 120 gatgattata acttaaaatg gtttttatttcctattgatg atgatcaata tattattaca 180 agctatgcag caaataattg taaagtttggaatgttaata atgataaaat aaatgtttcg 240 acttattctt caacaaattc aatacaaaaatggcaaataa aagctaatgg ttcttcatat 300 gtaatacaaa gtgataatgg aaaagtcttaacagcaggaa ccggtcaagc tcttggattg 360 atacgtttaa ctgatgaatc ctcaaataatcccaatcaac aatggaattt aacttctgta 420 caaacaattc aacttccaca aaaacctataatagatacaa aattaaaaga ttatcccaaa 480 tattcaccaa ctggaaatat agataatggaacatctcctc aattaatggg atggacatta 540 gtaccttgta ttatggtaaa tgatccaaatatagataaaa atactcaaat taaaactact 600 ccatattata ttttaaaaaa atatcaatattggcaacgag cagtaggaag taatgtagct 660 ttacgtccac atgaaaaaaa atcatatacttatgaatggg gcacagaaat agatcaaaaa 720 acaacaatta taaatacatt aggatttcaaatcaatatag attcaggaat gaaatttgat 780 ataccagaag taggtggagg tacagatgaaataaaaacac aactaaatga agaattaaaa 840 atagaatata gtcatgaaac taaaataatggaaaaatatc aagaacaatc tgaaatagat 900 aatccaactg atcaatcaat gaattctataggatttctta ctattacttc cttagaatta 960 tatagatata atggctcaga aattcgtataatgcaaattc aaacctcaga taatgatact 1020 tataatgtta cttcttatcc aaatcatcaacaagctttat tacttcttac aaatcattca 1080 tatgaagaag tagaagaaat aacaaatattcctaaaagta cactaaaaaa attaaaaaaa 1140 tattatttta tgtcagcacg tgaagtacacattgatgtaa ataataagac aggtcataca 1200 ttacaattag aagataaaac aaaacttgatggtggtagat ggcgaacatc acctacaaat 1260 gttgctaatg atcaaattaa aacatttgtagcagaatcaa atggttttat gacaggtaca 1320 gaaggtacta tatattatag tataaatggagaagcagaaa ttagtttata ttttgacaat 1380 ccttttgcag gttctaataa atatgatggacattccaata aatctcaata tgaaattatt 1440 acccaaggag gatcaggaaa tcaatctcatgttacgtata ctattcaaac cacatcctca 1500 cgatatgggc ataaatcata a 1521 16123 DNA Artificial Sequence Description of Artificial Sequence primer45kD5′ 161 gatratratc aatatattat tac 23 162 20 DNA Artificial SequenceDescription of Artificial Sequence primer 45kD3′rc 162 caaggtartaatgtccatcc 20 163 24 DNA Artificial Sequence Description of ArtificialSequence primer 45kD5′01 163 gatgatgrtm rakwwattat trca 24 164 24 DNAArtificial Sequence Description of Artificial Sequence primer 45kD5′02164 gatgatgrtm ratatattat trca 24 165 23 DNA Artificial SequenceDescription of Artificial Sequence primer 45kD3′03 165 ggawgkrcdytwdtmccwtg tat 23 166 23 DNA Artificial Sequence Description ofArtificial Sequence primer 45kD3′04 166 ggawgkacry tadtaccttg tat 23

What is claimed is:
 1. An isolated polynucleotide that encodes a fusionprotein comprising a first amino acid sequence and a second amino acidsequence, wherein said first amino acid sequence is the amino acidsequence of an approximately 45 kDa polypeptide and said second aminoacid sequence is the amino acid sequence of an approximately 15 kDapolypeptide, wherein said polypeptides are toxic to a rootworm pest thatingests said polypeptides, wherein a nucleotide sequence that codes forsaid first amino acid sequence hybridizes with the complement of thenucleic acid sequence of SEQ ID NO:10, and wherein a nucleotide sequencethat codes for said second amino acid sequence hybridizes with thecomplement of the nucleic acid sequence of SEQ ID NO:31.
 2. An isolatedpolynucleotide that encodes a fusion protein comprising a first aminoacid sequence and a second amino acid sequence wherein said fusionprotein is toxic to a rootworm pest that ingests said protein; whereinsaid first amino sequence is the amino sequence of an approximately 45kDa polypeptide and said second amino acid sequence is the amino acidsequence of an approximately 15 kDa polypeptide; wherein a nucleotidesequence that codes for said first amino acid sequence hybridizes understringent conditions with the complement of a nucleic acid sequenceselected form the group consisting of SEQ ID NO:10, SEQ ID NO:42, andSEQ ID NO:45; and wherein a nucleotide sequences that codes for saidsecond amino acid sequence hybridizes under stringent condition with thecomplement of nucleic acid sequence selected from the group consistingof SEQ ID NO:31, SEQ ID NO:40, and SEQ ID NO:44.
 3. The polynucleotideaccording to claim 1 wherein said polynucleotide encodes a fusionprotein comprising the amino acid sequence of SEQ ID NO:159.
 4. Thepolynucleotide according to claim 1 wherein said polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:160.
 5. Thepolynucleotide according to claim 2 wherein a nucleotide sequence thatcodes for said first amino acid sequence hybridizes under stringentconditions with the complement of the nucleic acid sequence of SEQ IDNO:10.
 6. The polynucleotide according to claim 2 wherein a nucleotidesequence that codes for said second amino acid sequence hybridizes understringent conditions with the complement of the nucleic acid sequence ofSEQ ID NO:31.
 7. The polynucleotide according to claim 2 wherein anucleotide sequence that codes for said first amino acid sequencehybridizes under stringent conditions with the complement of the nucleicacid sequence of SEQ ID NO:42.
 8. The polynucleotide according to claim2 wherein a nucleotide sequence that codes for said second amino acidsequence hybridizes under stringent conditions with the complement ofthe nucleic acid sequence of SEQ ID NO:40.
 9. The polynucleotideaccording to claim 2 wherein a nucleotide sequence that codes for saidfirst amino acid sequence hybridizes under stringent conditions with thecomplement of the nucleic acid sequence SEQ ID NO:45.
 10. Thepolynucleotide according to claim 2 wherein a nucleotide sequence thatcodes for said amino acid sequence hybridizes under stringent conditionswith the complement of the nucleic acid sequence of SEQ ID NO:44. 11.The polynucleotide according to claim 2 wherein said polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:44.
 12. Thepolynucleotide according of claim 2 wherein said polynucleotidecomprises the nucleic acid sequence of SEQ ID NO:45.
 13. Thepolynucleotide according to claim 2 wherein said first amino acidsequence is SEQ ID NO:11.
 14. The polynucleotide according to claim 2wherein said second amino acid sequence is SEQ ID NO:32.
 15. Thepolynucleotide according to claim 2 wherein said first amino acidsequence is SEQ ID NO:43.
 16. The polynucleotide according to claim 2wherein said second amino acid sequence is SEQ ID NO:41.
 17. Thepolynucleotide of claim 1 wherein said polynucleotide comprises a firstsegment and a second segment, wherein said first segment encodes saidfirst amino acid sequence and said second segment encodes said secondamino acid sequence, and wherein said second segment is 5′ to said firstsegment.
 18. The polynucleotide of claim 1 wherein said second aminoacid sequence is at the carboxy terminus of said protein and said firstamino acid sequence is at the amino terminus of said protein.
 19. Atransgenic host cell comprising a polynucleotide of claim 1, whereinsaid cell is selected from the group consisting of a plant cell and abacterial cell.
 20. The polynucleotide according to claim 2, whereinsaid first amino acid sequence is SEQ ID NO:11.
 21. The polynucleotideaccording to claim 2 wherein said second amino acid sequence is SEQ IDNO:32.
 22. The polynucleotide of claim 2 wherein said polynucleotidecomprises a first segment and a second segment, wherein said firstsegment encodes said first amino aid sequence and said second segmentencodes said second amino acid sequence, and wherein said second segmentis 5′ to said first segment.
 23. The polynucleotide of claim 2 whereinsaid second amino acid sequence is at the carboxy terminus of saidprotein and said first amino acid sequence is at the amino terminus ofsaid protein.
 24. A transgenic host cell comprising a polynucleotide ofclaim 2, wherein said cell is selected from the group consisting of aplant cell and a bacterial cell.